Genetic sequences encoding flavonoid pathway enzymes and uses therefor

ABSTRACT

PCT No. PCT/AU93/00127 Sec. 371 Date Nov. 30, 1994 Sec. 102(e) Date Nov. 30, 1994 PCT Filed Mar. 25, 1993 PCT Pub. No. WO93/20206 PCT Pub. Date Oct. 14, 1993The present invention relates to a nucleic acid isolate comprising a sequence of nucleotides encoding, or complementary to a sequence encoding, a flavonoid 3&#39;-hydroxylase or a functional derivative thereof. The present invention also relates to transgenic plants carrying and/or expressing the above-mentioned nucleic acid material.

The present invention relates generally to genetic sequences encodingflavonoid pathway metabolising enzymes and more particularly toflavonoid 3'-hydroxylase or fragments or derivatives thereof and theiruse in the manipulation of pigmentation in plants and other organisms.

The flower industry strives to develop new and different varieties offlowering plants. An effective way to create such novel varieties isthrough the manipulation of flower colour. Classical breeding techniqueshave been used with some success to produce a wide range of colours formost of the commercial varieties of flowers. This approach has beenlimited, however, by the constraints of a particular species' gene pooland for this reason it is rare for a single species to have a fullspectrum of coloured varieties. In addition, traditional breedingtechniques lack precision. The aesthetic appeal of the flower is acombination of many factors such as form, scent and colour; modificationof one character through hybridization can often be at the expense of anequally valuable feature. The ability to engineer precise colour changesin cutflower and ornamental species would offer significant commercialopportunities in an industry which has rapid product turnover and wherenovelty is an important market characteristic.

Flower colour is predominantly due to two types of pigment: flavonoidsand carotenoids. Flavonoids contribute to a range of colours from yellowto red to blue. Carotenoids impart an orange or yellow tinge and arecommonly the major pigment in yellow or orange flowers. The flavonoidmolecules which make the major contribution to flower colour are theanthocyanins which are glycosylated derivatives of cyanidin,delphinidin, petunidin, peonidin, malvidin and pelargonidin, and arelocalised in the vacuole. The different anthocyanins can produce markeddifferences in colour. Flower colour is also influenced byco-pigmentation with colourless flavonoids, metal complexation,glycosylation, acylation and vacuolar pH (Forkmann, 1991).

The biosynthetic pathway for the flavonoid pigments (hereinafterreferred to as the "flavonoid pathway") is well established and is shownin FIG. 1 (Ebel and Hahlbrock, 1988; Hahlbrock and Grisebach, 1979;Wiering and De Vlaming, 1984; Schram et al., 1984; Stafford, 1990). Thefirst committed step in the pathway involves the condensation of threemolecules of malonyl-CoA with one molecule of p-coumaroyl-CoA. Thisreaction is catalysed by the enzyme chalcone synthase (CHS). The productof this reaction, 2',4,4',6', tetrahydroxy-chalcone, is normally rapidlyisomerized to produce naringenin by the enzyme chalcone flavanoneisomerase (CHI). Naringenin is subsequently hydroxylated at the 3position of the central ring by flavanone 3-hydroxylase (F3H) to producedihydrokaempferol (DHK).

The pattern of hydroxylation of the B-ring of DHK plays a key role indetermining petal colour. The B-ring can be hydroxylated at either the3', or both the 3' and 5' positions, to produce dihydroquercetin (DHQ)and dihydromyricetin (DHM), respectively. Two key enzymes involved inthis pathway are flavonoid 3'-hydroxylase and flavonoid3',5'-hydroxylase, both of the cytochrome P450 class. Cytochrome P450enzymes are widespread in nature and genes have been isolated andsequenced from vertebrates, insects, yeasts, fungi, bacteria and plants.

Flavonoid 3'-hydroxylase acts on DHK to produce DHQ and on naringenin toproduce eriodictyol. Reduction and glycosylation of DHQ produces thecyanidin-glycoside and peonidin-glycoside pigments which, in many plantspecies (for example rose, carnation and chrysanthemum), contribute tored and pink flower colour. The synthesis of these anthocyanins can alsoresult in other flower colours. For example, blue cornflowers containcyanin. The ability to control flavonoid 3'-hydroxylase activity, orother enzymes involved in the flavonoid pathway, in flowering plantswould provide a means to manipulate petal colour. Different colouredversions of a single cultivar could thereby be generated and in someinstances a single species would be able to produce a broader spectrumof colours.

In accordance with the present invention, the genetic sequences encodingflavonoid 3'-hydroxylase have been identified and cloned. Theserecombinant sequences permit the modulation of hydroxylation ofsubstrates such as DHK and naringenin, leading to a modification ofanthocyanin composition, thereby providing a means to manipulate petalcolour. The presence of the flavonoid 3'-hydroxylase would allow thediversion of the metabolic pathway from DHK to anthocyanin derivativesof anthocyanidins such as cyanidin and peonidin, thereby providing ameans to manipulate petal colour by modulation of the level of3'-hydroxylation. Accordingly, the present invention relates to thealtering of flavonoid 3'-hydroxylase activity in plants, whichencompasses elevating or reducing levels of existing flavonoid3'-hydroxylase activity by introducing the sequences of the presentinvention. Reduction in levels of flavonoid 3'-hydroxylase activity mayalso be referred to as down-regulation. Moreover, the present inventionextends beyond flowers to fruit and vegetable plants and to leaves of,for example, ornamental plants.

Accordingly, one aspect of the present invention provides a nucleic acidisolate comprising a sequence of nucleotides encoding, or complementaryto a sequence encoding, flavonoid 3'-hydroxylase enzyme (hereinafterreferred to as 3'-hydroxylase) or a functional derivative of the enzyme.

By the term "nucleic acid isolate" is meant a genetic sequence in anon-naturally-occurring condition. Generally, this means isolated awayfrom its natural state or synthesized or derived in anon-naturally-occurring environment. More specifically, it includesnucleic acid molecules formed or maintained in vitro, including genomicDNA fragments, recombinant or synthetic molecules and nucleic acids incombination with heterologous nucleic acids. It also extends to thegenomic DNA or cDNA or part thereof encoding 3'-hydroxylase or partthereof in reverse orientation relative to its or another promoter. Itfurther extends to naturally-occurring sequences following at least apartial purification relative to other nucleic acid sequences.

The term "genetic sequences" is used herein in its most general senseand encompasses any contiguous series of nucleotide bases specifyingdirectly, or via a complementary series of bases, a sequence of aminoacids in a 3'-hydroxylase. Such a sequence of amino acids may constitutea full-length 3'-hydroxylase or an active truncated form thereof or maycorrespond to a particular region such as an N-terminal, C-terminal orinternal portion of the enzyme. The nucleic acid sequences contemplatedherein also encompass oligonucleotides useful as genetic probes or as"antisense" molecules capable of regulating expression of thecorresponding gene in a plant. An "antisense molecule" as used hereinmay also encompass a gene construct comprising the structural genomic orcDNA gene or part thereof in reverse orientation relative to its oranother promoter.

In one embodiment the nucleic acid sequence encoding 3'-hydroxylase orvarious functional derivatives thereof are used to reduce the activityof an endogenous 3'-hydroxylase, or alternatively the nucleic acidsequence encoding this enzyme or various derivatives or parts thereofare used in the antisense orientation to reduce activity of the3'-hydroxylase. Although not wishing to limit the present invention toany one theory, it is possible that an antisense 3'-hydroxylasetranscript or fragment or part thereof (for example, an oligonucleotidemolecule) would form a duplex with all or part of the naturallyoccurring mRNA specified for the enzyme thus preventing accumulation ofor translation from the mRNA into active enzyme. In a furtheralternative, ribozymes could be used to inactivate target nucleic acidsequences.

Reference herein to the altering of flavonoid 3'-hydroxylase activityrelates to an elevation or reduction in activity of up to 30% or morepreferably of 30-50%, or even more preferably 50-75% or still morepreferably 75% or greater above or below the normal endogenous orexisting levels of activity. The level of activity can be readilyassayed using a modified version of the method described by Stotz andForkmann (1982) (see Example 1).

The nucleic acids of the present invention may be a ribonucleic acid ordeoxyribonucleic acids, single or double stranded and linear orcovalently closed circular molecules. Preferably, the nucleic acidmolecule is cDNA. The present invention also extends to other nucleicacid molecules which hybridize under low, preferably under medium andmost preferably under high stringency conditions with the nucleic acidmolecules of the present invention and in particular to the sequence ofnucleotides set forth in FIG. 5 or a part or region thereof. In its mostpreferred embodiment, the present invention extends to a nucleic acidmolecule having a nucleotide sequence set forth in FIG. 5 or to amolecule having at least 40%, more preferably at least 45%, even morepreferably at least 55%, still more preferably at least 65-70%, and yeteven more preferably greater than 85% similarity at the level ofnucleotide or amino acid sequence to at least one or more regions of thesequence set forth in FIG. 5 and wherein the nucleic acid encodes or iscomplementary to a sequence which encodes an enzyme having3'-hydroxylase activity. It should be noted, however, that nucleotide oramino acid sequences may have similarities below the above givenpercentages and yet still encode 3'-hydroxylase activity and suchmolecules may still be considered in the scope of the present inventionwhere they have regions of sequence conservation. The present inventionfurther extends to nucleic acid molecules in the form of oligonucleotideprimers or probes capable of hybridizing to a portion of the nucleicacid molecules contemplated above, and in particular those set forth inFIG. 5, under low, preferably under medium and most preferably underhigh stringency conditions. Preferably the portion corresponds to the 5'or the 3' end of the gene. For convenience the 5' end is consideredherein to define a region substantially between the start codon of thestructural genetic sequence to a centre portion of the gene, and the 3'end is considered herein to define a region substantially between thecentre portion of the gene and the terminating codon of the structuralgenetic sequence. It is clear, therefore, that oligonucleotides orprobes may hybridize to the 5' end or the 3' end or to a region commonto both the 5' and the 3' ends. The present invention extends to allsuch probes. Preferred oligonucleotides are set forth in Example 1.

The nucleic acid or its complementary form may encode the full-lengthenzyme or a part or derivative thereof. By "derivative" is meant anysingle or multiple amino acid substitutions, deletions, and/or additionsrelative to the naturally-occurring enzyme and which retains3'-hydroxylase activity. In this regard, the nucleic acid includes thenaturally-occurring nucleotide sequence encoding 3'-hydroxylase or maycontain single or multiple nucleotide substitutions, deletions and/oradditions to said naturally-occurring sequence. The nucleic acid of thepresent invention or its complementary form may also encode a "part" ofthe 3'-hydroxylase, whether active or inactive, and such a nucleic acidmolecule may be useful as an oligonucleotide probe, primer forpolymerase chain reactions or in various mutagenic techniques, or forthe generation of antisense molecules.

Amino acid insertional derivatives of the 3'-hydroxylase of the presentinvention include amino and/or carboxyl terminal fusions as well asintra-sequence insertions of single or multiple amino acids. Insertionalamino acid sequence variants are those in which one or more amino acidresidues are introduced into a predetermined site in the proteinalthough random insertion is also possible with suitable screening ofthe resulting product. Deletional variants are characterised by theremoval of one or more amino acids from the sequence. Substitutionalamino acid variants are those in which at least one residue in thesequence has been removed and a different residue inserted in its place.Typical substitutions are those made in accordance with Table 1overleaf.

Where the 3'-hydroxylase is derivatised by amino acid substitution, theamino acids are generally replaced by other amino acids having likeproperties, such as hydrophobicity, hydrophilicity, electronegativity,bulky side chains and the like. Amino acid substitutions are typicallyof single residues. Amino acid insertions will usually be in the orderof about 1-10 amino acid residues and deletions will range from about1-20 residues. Preferably, deletions or insertions are made in adjacentpairs, i.e. a deletion of two residues or insertion of two residues.

The amino acid variants referred to above may readily be made usingpeptide synthetic techniques well known in the art, such as solid phasepeptide synthesis (Merrifield, 1964) and the like, or by recombinant DNAmanipulations. Techniques for making substitution mutations atpredetermined sites in DNA having known or partially known sequence arewell known and include, for example, M13 mutagenesis. The manipulationof DNA sequence to produce variant proteins which manifest assubstitutional, insertional or deletional variants are convenientlydescribed, for example, in Sambrook et al. (1989).

                  TABLE 1                                                         ______________________________________                                        Suitable residues for amino acid substitutions                                Original Residue  Exemplary Substitutions                                     ______________________________________                                        Ala               Ser                                                         Arg               Lys                                                         Asn               Gln; His                                                    Asp               Glu                                                         Cys               Ser                                                         Gln               Asn                                                         Glu               Asp                                                         Gly               Pro                                                         His               Asn; Gln                                                    Ile               Leu; Val                                                    Leu               Ile; Val                                                    Lys               Arg; Gln; Glu                                               Met               Leu; Ile                                                    Phe               Met; Leu; Lyr                                               Ser               Thr                                                         Thr               Ser                                                         Trp               Tyr                                                         Tyr               Trp; Phe                                                    Val               Ile; Leu                                                    ______________________________________                                    

Other examples of recombinant or synthetic mutants and derivatives ofthe 3'-hydroxylase of the present invention include single or multiplesubstitutions, deletions and/or additions of any molecule associatedwith the enzyme such as carbohydrates, lipids and/or proteins orpolypeptides.

The terms "analogues" and "derivatives" also extend to any functionalchemical equivalent of the 3'-hydroxylase and also to any amino acidderivative described above. For convenience, reference to"3'-hydroxylase" herein includes reference to any mutants, derivatives,analogues, homologues or fragments thereof.

The present invention is exemplified using nucleic acid sequencesderived from petunia since this represents the most convenient andpreferred source of material to date. However, one skilled in the artwill immediately appreciate that similar sequences can be isolated fromany number of sources such as other plants or certain microorganisms.Examples of other plants include, but are not limited to, carnation,chrysanthemum, rose, maize, snapdragon, tobacco, cornflower, pelargoniumand morning glory. All such nucleic acid sequences encoding directly orindirectly a flavonoid pathway enzyme and in particular 3'-hydroxylase,regardless of their source, are encompassed by the present invention.

The nucleic acid molecules contemplated herein may exist in eitherorientation alone or in combination with a vector molecule, for examplean expression-vector. The term vector molecule is used in its broadestsense to include any intermediate vehicle for the nucleic acid molecule,capable of facilitating transfer of the nucleic acid into the plant celland/or facilitating integration into the plant genome. An intermediatevehicle may, for example, be adapted for use in electroporation,microprojectile bombardment, Agrobacterium-mediated transfer orinsertion via DNA or RNA viruses. The intermediate vehicle and/or thenucleic acid molecule contained therein may or may not need to be stablyintegrated into the plant genome. Such vector molecules may alsoreplicate and/or express in prokaryotic cells. Preferably, the vectormolecules or parts thereof are capable of integration into the plantgenome. The nucleic acid molecule may additionally contain a promotersequence capable of directing expression of the nucleic acid molecule ina plant cell. The nucleic acid molecule and promoter may also beintroduced into the cell by any number of means such as those describedabove.

In accordance with the present invention, a nucleic acid sequenceencoding 3'-hydroxylase or a derivative or part thereof may beintroduced into and expressed in a plant in either orientation therebyproviding a means either to convert DHK and/or other suitablesubstrates, if synthesised in the plant cell, ultimately intoanthocyanin derivatives of anthocyanidins such as cyanidin and/orpeonidin, or alternatively to inhibit such conversion of metabolites byreducing or eliminating endogenous or existing 3'-hydroxylase activity.The production of anthocyanins contributes to the production of a red orblue flower colour. Expression of the nucleic acid sequence in eitherorientation in the plant may be constitutive, inducible ordevelopmental, and may also be tissue-specific. The word expression isused in its broadest sense to include production of RNA or of both RNAand protein. It also extends to partial expression of a nucleic acidmolecule.

According to this aspect of the present invention there is provided amethod for producing a transgenic plant capable of synthesizing3'-hydroxylase or active mutants or derivatives thereof, said methodcomprising stably transforming a cell of a suitable plant with a nucleicacid molecule which comprises a sequence of nucleotides encoding said3'-hydroxylase, under conditions permitting the eventual expression ofsaid nucleic acid molecule, regenerating a transgenic plant from thecell and growing said transgenic plant for a time and under conditionssufficient to permit the expression of the nucleic acid. The transgenicplant may thereby produce elevated levels of 3'-hydroxylase activityrelative to the amount expressed in a comparable non-transgenic plant.

Another aspect of the present invention contemplates a method forproducing a transgenic plant with reduced endogenous or existing3'-hydroxylase activity, said method comprising stably transforming acell of a suitable plant with a nucleic acid molecule which comprises asequence of nucleotides encoding or complementary to a sequence encoding3'-hydroxylase, regenerating a transgenic plant from the cell and wherenecessary growing said transgenic plant under conditions sufficient topermit the expression of the nucleic acid.

Yet another aspect of the present invention contemplates a method forproducing a genetically modified plant with reduced endogenous orexisting 3'-hydroxylase activity, said method comprising altering the3'-hydroxylase gene through modification of the endogenous sequences viahomologous recombination from an appropriately altered 3'-hydroxylasegene or derivative or part thereof introduced into the plant cell, andregenerating the genetically modified plant from the cell.

In a preferred embodiment, the present invention contemplates a methodfor producing a transgenic flowering plant exhibiting alteredinflorescence properties, said method comprising stably transforming acell of a suitable plant with a nucleic acid sequence of the presentinvention, regenerating a transgenic plant from the cell and growingsaid transgenic plant for a time and under conditions sufficient topermit the expression of the nucleic acid sequence into the3'-hydroxylase enzyme. Alternatively, said method may comprise stablytransforming a cell of a suitable plant with a nucleic acid sequence ofthe present invention or its complementary sequence, regenerating atransgenic plant from the cell and growing said transgenic plant for atime and under conditions sufficient to alter the level of activity ofthe endogenous or existing 3'-hydroxylase. Preferably the altered levelwould be less than the endogenous or existing level of 3'-hydroxylaseactivity in a comparable non-transgenic plant. Without wishing to limitthe present invention, one theory of mode of action is that reduction ofthe endogenous 3'-hydroxylase activity requires the expression of theintroduced nucleic acid sequence or its complementary sequence. However,expression of the introduced genetic sequence or its complement may notbe required to achieve the desired effect: namely, a flowering plantexhibiting altered inflorescence properties.

In a related embodiment, the present invention contemplates a method forproducing a flowering plant exhibiting altered inflorescence properties,said method comprising alteration of the 3'-hydroxylase gene throughmodification of the endogenous sequences via homologous recombinationfrom an appropriately altered 3'-hydroxylase gene or derivative or partthereof introduced into the plant cell, and regenerating the geneticallymodified plant from the cell.

The nucleic acid molecule of the present invention may or may not bedevelopmentally regulated. Preferably, the altered inflorescenceincludes the production of red flowers or other colour shades dependingon the physiological conditions of the recipient plant. By "recipientplant" is meant a plant capable of producing a substrate for the3'-hydroxylase enzyme, or producing the 3'-hydroxylase enzyme itself,and possessing the appropriate physiological properties and genotyperequired for the development of the colour desired. This may include butis not limited to petunia, carnation, chrysanthemum, rose, snapdragon,tobacco, cornflower, pelargonium, lisianthus and morning glory.

Accordingly, the present invention extends to a method for producing atransgenic plant capable of expressing a recombinant gene encoding3'-hydroxylase or part thereof or which carries a nucleic acid sequencewhich is substantially complementary to all or a part of a mRNA moleculeoptionally transcribable where required to effect regulation of a3'-hydroxylase, said method comprising stably transforming a cell of asuitable plant with the nucleic acid isolate comprising a sequence ofnucleotides encoding, or complementary to a sequence encoding,3'-hydroxylase or a derivative or part thereof, where necessary underconditions permitting the eventual expression of said nucleic acidisolate, and regenerating a transgenic plant from the cell.

One skilled in the art will immediately recognise the variationsapplicable to the methods of the present invention, such as increasingor decreasing the expression of the enzyme naturally present in a targetplant leading to differing shades of colours such as different shades ofred.

The present invention, therefore, extends to all transgenic plantscontaining all or part of the nucleic acid sequence of the presentinvention and/or any homologues or related forms thereof or antisenseforms of any of these and in particular those transgenic plants whichexhibit altered inflorescence properties. The transgenic plants maycontain an introduced nucleic acid molecule comprising a nucleotidesequence encoding or complementary to a sequence encoding3'-hydroxylase. Generally the nucleic acid would be stably introducedinto the plant genome, although the present invention also extends tothe introduction of the 3'-hydroxylase nucleotide sequence within anautonomously-replicating nucleic acid sequence such as a DNA or RNAvirus capable of replicating within the plant cell. The invention alsoextends to seeds from such transgenic plants. Such seeds, especially ifcoloured, will be useful as proprietary tags for plants.

A further aspect of the present invention is directed to recombinantforms of 3'-hydroxylase. The recombinant forms of the enzymes willprovide a source of material for research to develop, for example, moreactive enzymes and may be useful in developing in vitro systems forproduction of coloured compounds.

Still a further aspect of the present invention contemplates the use ofthe genetic sequences described herein in the manufacture of a geneticconstruct capable of expressing a 3'-hydroxylase enzyme ordown-regulating an endogenous 3'-hydroxylase in a plant.

The present invention is further described by reference to the followingnon-limiting Figures and Example.

In the Figures:

FIGS. 1A and B are is a schematic representation of the biosynthesispathway for the flavonoid pigments. Enzymes involved in the first partof the pathway have been indicated as follows: PAL=Phenylalanineammonia-lyase; C4H=Cinnamate 4-hydroxylase; 4CL=4-coumarate: CoA ligase;CHS=Chalcone synthase; CHI=Chalcone flavanone isomerase; F3H=Flavanone3-hydroxylase; DFR=Dihydroflavonol-4-reductase; UFGT=UDP-glucose:flavonoid-3-O-glucosyl-transferase. The later steps correspond toconversions that occur in P. hybrida flowers and include: 1=addition ofa rhamnose sugar to the glucosyl residue of cyanidin-3-glucoside anddelphinidin-3-glucoside; 2=acylation and 5-O-glucosylation; 3=3'methylation; 4=5' methylation; 5=3',5' methylation.

FIG. 2(A) is a schematic representation of DNA fragments used to probecDNA library #1 to identify cytochrome P450 homologues. P450:generalized cytochrome P450 cDNA clone with the haem-binding domain(Haem) indicated by the shaded box; pCGP142: a 980 bp fragment wasobtained by PCR with oligos 1 and 2 using pCGP142 DNA as template;pCGP147: a 1.3 kb fragment was isolated from a SalI-EcoRI digest ofpCGP147; pCGP158: a 900 bp fragment was obtained by PCR with oligos 3and 4 using pCGP158 DNA as template; pCGP160: a 600 bp fragment wasisolated from a PstI-EcoRV digest of pCGP160; pCGP454: fragment wasobtained by PCR with oligos 3 and 5 using pCGP454 DNA as template. Allpurified fragments were labelled with ³² P-dCTP as described in Example1.

FIGS. 2(B) to (H) show partial nucleotide sequences and thecorresponding predicted amino acid translation products for the cDNAinserts from (i) pCGP142 (SEQ ID NOS:27, 28), (ii) pCGP147 (SEQ IDNOS:29,30), (iii) pCGP158 (SEQ ID NOS:31,32), (iv) pCGP160 (SEQ IDNOS:33,34) and (v) pCGP454 (SEQ ID NOS:35,36). The regions used to probecDNA library #1 to isolate related clones have been delineated byarrowheads.

FIGS. 3(A) to (D) is the nucleotide sequence (SEQ ID NO:37) andpredicted amino acid sequence (SEQ ID NO:38) for the cDNA insert frompCGP602. Two probes that included the sequences between the internalHincII-EcoRV and EcoRV-HindIII sites were used to identify relatedsequences in a group of cytochrome P450 homologues.

FIGS. 4(A) and 4(B) show partial nucleotide sequence for the cDNAinserts from: 4(A): 1) pCGP161 (SEQ ID NO:39); 2) pCGP162 (SEQ IDNO:40); 3) pCGP163 (SEQ ID NO:41); 4) pCGP165 (SEQ ID NO:42); 5) pCGP166(SEQ ID NO:43); 6) pCGP167 (SEQ ID NO:44), and 4(B): 7 pCGP168 (SEQ IDNO:45); 8) pCGP169 (SEQ ID NO:46); 9) pCGP171 (SEQ ID NO:47) and 10)pCGP173 (SEQ ID NO:48). A mixed probe that included the cDNA inserts ofall these clones was used to screen cDNA library #2 for relatedsequences.

FIGS. 5A-D are the nucleotide sequence (SEQ ID NO:49) and predictedamino acid sequence (SEQ ID NO:50) for the cDNA insert from pCGP619.

FIG. 6 shows a diagrammatic representation of a restriction enzyme mapof pCGP619. Partial lengths of the cDNA insert are indicated by thebolder lines with solid ends (as opposed to arrows). These weresubcloned into M13-mp18 and mp19 and sequenced using oligonucleotideprimer sequences, as indicated, to obtain overlapping sequenceinformation. The extent and direction of sequence information obtainedfrom each subcloned piece is shown by lines with arrowheads. Primer -40was used unless otherwise specified. 190=primer sequence 190; 191=primersequence 191; poly T=poly T oligonucleotide was used as primer; dsseq=sequence was read with double-stranded DNA; ATG indicates themethionine initiation codon and the total length of the clone in basepairs is also indicated.

FIG. 7 shows a 3'-hydroxylase assay of yeast extracts using ³H-naringenin as substrate. The autoradiograph shows conversion of ³H-naringenin to the 3'-hydroxylated derivative eriodictyol by an extractof yeast transformed with the plasmid pCGP621 (1, 2). No 3'-hydroxylaseactivity was detected in untransformed yeast (C).

FIG. 8 shows nucleotide sequence (SEQ ID NO:51) and predicted amino acidsequence (SEQ ID NO:52) for the insert from pCGP635. These sequences maybe used as probes for the isolation of putative rose 3'-hydroxylase cDNAclones.

FIGS. 9A and B show nucleotide sequence (SEQ ID NO:53) and predictedamino acid sequence (SEQ ID NO:54) for the insert from pCGP772. Thesesequences may be used as probes for the isolation of putative carnation3'-hydroxylase cDNA clones.

FIGS. 10A and B show nucleotide sequence (SEQ ID NO:55) and predictedamino acid sequence (SEQ ID NO:56) for the insert from pCGP773. Thesesequences may be used as probes for the isolation of carnation putative3'-hydroxylase cDNA clones.

FIG. 11 shows partial nucleotide sequence (SEQ ID NO:57) and predictedamino acid sequence (SEQ ID NO:58) for insert from pCGP854. Thesesequences were used as a probe to select a putative 3'-hydroxylase cDNAclone. Underlined amino acids are identical to those of the cDNA insertfrom pCGP619 between positions 971 and 1091.

The disarmed microorganism Agrobacterium tumefaciens strain AGL0containing the plasmid pCGP809 was deposited with the AustralianGovernment Analytical Laboratories, 1 Suakin Street, Pymble, New SouthWales, 2037, Australia on Mar. 24, 1993 and was given Accession Number

EXAMPLE 1 Isolation of 3'-Hydroxylase and Related Nucleic AcidSequences 1. Materials and Methods

Chemicals Enzymes and Radioisotopes

Eriodictyol was obtained from Carl Roth KG and naringenin was obtainedfrom Sigma. [³ H]-Naringenin (5.7 Ci/mmole) was obtained from Amersham.All enzymes were obtained from commercial sources and used according tothe manufacturer's recommendations.

Bacterial Strains

The Escherichia coli strains used were:

DH5α supE44, Δ(lacZYA-ArgF)U169, φ80lacZΔM15, hsdR17 (r_(k) -, m_(k) +),

recA1, endA1, gyrA96, thi-1, relA1, deoR. (Hanahan, 1983 and BRL, 1986).

XL1-Blue supE44, hsdR17 (r_(k) -, m_(k) +), recA1, endA1, gyrA96, thi-1,relA1,

lac-, [F'proAB, lacI^(q), lacZΔM15, Tn10(tet^(r))] (Bullock et al.,1987).

PLK-F' recA, hsdR17 (r_(k) -, m_(k) +), mcrA⁻, mcrB⁻, lac⁻, supE44,galK2, galT22,

metB1, [F' proAB, lacI^(q), lacZΔM15, Tn10 (tet^(r))] (Stratagene).

SOLR e14⁻ (mcrA), Δ(mcrCB-hsdSMR-mrr)171, sbcC, recB, recJ,

umuC::Tn5(kan^(r)), uvrC,lac, gyrA96, thi-1, relA1, [F'proAB,

lacI^(q) ZΔM15], Su⁻ (non-suppressing) (Stratagene)

The disarmed Agrobacterium tumefaciens strain AGL0 (Lazo et al., 1991)was obtained from R Ludwig (Department of Biology, University ofCalifornia, Santa Cruz).

The cloning vector pBluescript was obtained from Stratagene.

Transformation of E. coli and A. tumefaciens

Transformation of the E. coli strain DH5a cells was performed accordingto the method of Inoue et al. (1990).

The plasmid pCGP809 was introduced into the Agrobacterium tumefaciensstrain AGL0 by adding 5 mg of plasmid DNA to 100 mL of competent AGL0cells prepared by inoculating a 50 mL MG/L (Garfinkel and Nester, 1980)culture and growing for 16 h with shaking at 28° C. The cells were thenpelleted and resuspended in 0.5 mL of 85% (v/v) 100 mM CACl₂ /15% (v/v)glycerol. The DNA-Agrobacterium mixture was frozen by incubation inliquid N₂ for 2 min and then allowed to thaw by incubation at 37° C. for5 min. The DNA/bacterial mixture was then placed on ice for a further 10min. The cells were then mixed with 1 mL of MG/L media and incubatedwith shaking for 16 h at 28° C. Cells of A. tumefaciens carrying pCGP809were selected on MG/L agar plates containing 100 mg/mL gentamycin. Thepresence of pCGP809 was confirmed by Southern analysis of DNA isolatedfrom the gentamycin-resistant transformants.

Plant Material

Seed of the Petunia F₁ hybrid "Old Glory Blue" (OGB) was obtained fromBall Seed, USA.

Chrysanthemum morifolium cultivars were obtained from Baguley Flower andPlant Growers, Victoria.

Flowers of Dianthus caryophyllus cv. Laguna and Rosa hybrida cv.Kardinal were obtained from Van Wyk and Son Flower Supply, Victoria.

Plants were grown in specialised growth rooms with a 14 hr day length ata light intensity of 10,000 lux minimum and a temperature of 22° to 26°C.

Five stages of Petunia flower development were defined as follows:

    ______________________________________                                        Stage 1: Unpigmented, closed bud (<25 mm in length).                          Stage 2: Pigmented, closed bud (25-35 mm in length).                          Stage 3: Dark purple bud with emerging corolla (>35 mm                                 in length).                                                          Stage 4: Dark purple opened flower pre-anther dehiscence                               (>50 mm in length).                                                  Stage 5: Fully opened flower with all anthers dehisced.                       ______________________________________                                    

Stages of Chrysanthemum flower development were defined as follows:

    ______________________________________                                        Stage 0: No visible flower bud.                                               Stage 1: Flower bud visible: florets completely covered by                             the bracts.                                                          Stage 2: Flower buds opening: tips of florets visible.                        Stage 3: Florets tightly overlapped.                                          Stage 4: Tips of nearly all florets exposed; outer florets                             opening but none horizontal.                                         Stage 5: Outer florets horizontal.                                            Stage 6: Flower approaching maturity.                                         ______________________________________                                    

Stages of Dianthus caryophyllus flower development were defined asfollows:

    ______________________________________                                        Stage 1:                                                                              No visible flower bud.                                                Stage 2:                                                                              Flower buds opening: tips of florets visible.                         Stage 3:                                                                              Tips of nearly all florets exposed; outer florets opening,                    none horizontal.                                                      Stage 4:                                                                              Outer florets horizontal.                                             ______________________________________                                    

Stages of Rosa hybrida flower development were defined as follows:

    ______________________________________                                        Stage 1: Unpigmented, tightly closed bud (10-12 mm high;                               5 mm wide).                                                          Stage 2: Pigmented, tightly closed bud (15 mm high;                                    9 mm wide).                                                          Stage 3: Pigmented, closed bud; sepals just beginning to open                          (20-25 mm high; 13-15 mm wide)                                       Stage 4: Flower bud beginning to open; petals heavily                                  pigmented; sepals have separated (bud is 25-30 mm                             high and 18 mm wide).                                                Stage 5: Sepals completely unfolded; some curling.                                     Petals are heavily pigmented and unfolding (bud is                            30-33 mm high and 20 mm wide).                                       ______________________________________                                    

Construction of cDNA Library #1

Twenty grams of stage 3 to 4 Petunia cv. OGB flower limbs tissue washomogenised in 100 mL of PEB (200 mM Tris-HCl (pH 8.6), 60 mM KCl, 30 mMMgCl₂, 25 mM EGTA) containing 10 mM vanadyl ribonucleoside complex. Celldebris was removed by filtering the homogenate through sterile Miracloth(Calbiochem). The filtrate was layered on top of a step gradient of 6 mLof PEB containing 25% (w/v) sucrose, 250 units InhibitAce (5-Prime3-Prime), and 6 mL of PEB containing 50% (w/v) sucrose and 250 unitsInhibitAce in Ultra-Clear™ Quick-Seal™ (Beckman) centrifuge tubes. Thetubes were centrifuged for 3.5 h at 26,000 rpm in a 70 Ti rotor.Membrane-bound polysomes were collected from the 25% sucrose/50% sucroseinterface and added to a 4M guanidium isothiocyanate solution. RNA wasisolated from the denatured polysomes by pelleting through a 5.7M CsClcushion, as described by Turpen and Griffith (1986).

A Uni-ZAP™ XR vector kit (Stratagene) was used to construct adirectional cDNA library in λZAP using 25 μg of the polysomal RNA astemplate. The primary library, which contained 250,000 plaque formingunits (pfu), was amplified by overnight growth on NZY plates (Sambrooket al., 1989) and the amplified phage stock was eluted in PSB (100 mMNaCl, 8 mM MgSO₄, 50 mM Tris-HCl (pH 7.5), 0.01% (w/v) gelatin) asdescribed by Sambrook et al., (1989).

Construction of cDNA Library #2

Total RNA was isolated from the petal tissue of P. hybrida cv. OGB stage3 to 4 flowers using the method of Turpen and Griffith (1986). Poly(A)⁺RNA was selected from the total RNA by three cycles of oligo-dTcellulose chromatography (Aviv and Leder, 1972).

Two micrograms of poly(A)⁺ RNA were reverse transcribed in a 20 μLvolume containing 1×Superscript™ reaction buffer, 10 mM dithiothreitol(DTT), 500 μM dATP, 500 μM dGTP, 500 μM dTTP, 500 μM 5-methyl-dCTP, 0.75μg oligonucleotide #6 and 2 μL Superscript™ reverse transcriptase (BRL).The reaction mix was incubated at 37° C. for 50 min, 44° C. for 10 min,then placed on ice.

Second strand reaction mix (140 μL) was added to the first strandreaction. The second strand reaction mix consisted of 21 mM Tris-HCl,104 mM KCl, 5.3 mM MgCl₂, 171 μM β-NAD, 11.4 mM (NH₄)₂ SO₄, 214 μM dATP,642 μM dCTP, 214 μM dGTP, 214 μM dTTP, 4 mM DTT, 10 μCi ³² P-dCTP (3000Ci/mMole), 15 units E. coli DNA ligase, 40 units DNA polymerase(Boehringer) and 0.8 units RNAse H. The final mixture was incubated for150 min at 16° C. To make the double-stranded cDNA blunt-ended, 10 unitsT4 DNA polymerase was added, and the reaction continued for a further 15min at 16° C. The reaction was stopped and the cDNA purified byphenol/chloroform extraction, followed by chloroform extraction andethanol precipitation.

EcoRI adaptors (Promega) were ligated with the cDNA and then kinasedusing conditions recommended by the manufacturer. The enzymes weredenatured by heat (70° C., 20 min) and the DNA was purified byphenol/chloroform extraction and ethanol precipitation. The cDNA wasdigested with 50 units XhoI (Boehringer) in a reaction volume of 100 μL,using conditions recommended by the manufacturer. The enzyme was heatkilled (70° C., 20 min) and the mixture passed through an S400 spuncolumn (Pharmacia) which had been equilibrated in STE buffer (Sambrooket al., 1989).

The eluate was phenol/chloroform extracted and ethanol precipitated.After microcentrifugation at 4° C. for 30 min the cDNA pellet was rinsedwith 70% (v/v) ethanol, air dried and resuspended in 10 μL of TE buffer(10 mM Tris-HCl (pH 7.5), 1 mM EDTA).

NA-45 membrane (Schleicher and Schuell) was used to isolate cDNA in thesize range of 1.3 to 2.5 kb from a 7.5 μL sample that had beenelectrophoresed through a 1% (w/v) agarose gel.

The size fractionated cDNA was ligated with 1 μg λZAPII EcoRI/XhoI/CIAPtreated vector (Stratagene) in 5 μL reaction buffer consisting of 50 mMTris-HCl (pH 7.0), 10 mM MgCl₂, 10 mM DTT, 1 mM ATP and 2 units T4 DNAligase. The reaction was performed at 4° C. for 2 days.

After leaving at room temperature for 2 h, the ligation reaction mixturewas packaged using the Packagene system (Promega). The total number ofrecombinants was 270,000 pfu.

An amount of 150,000 pfu of the packaged cDNA was plated at 10,000 pfuper 15 cm diameter plate after transfecting PLK-F' cells. The plateswere incubated at 37° C. for 8 h, then stored overnight at 4° C.Duplicate lifts were taken onto Colony/Plaque Screen™ filters (DuPont)and treated as recommended by the manufacturer.

Construction of cDNA Library #3

Total RNA was isolated from the petal tissue of Chrysanthemum morifoliumcv. Dark Pink Pompom (Reference Number 5999), stages 1, 2 and 3 flowers,again using the method of Turpen and Griffith (1986). Poly(A)⁺ RNA wasselected from the total RNA, as for P. hybrida, by three cycles ofoligo-dT cellulose chromatography (Aviv and Leder, 1972). Two microgramsof poly(A)⁺ RNA were used as template for cDNA synthesis, as outlinedabove for P. hybrida.

Following fractionation and ligation, the cDNA reaction mixture waspackaged using the Packagene system (Promega). The total number ofrecombinants was 37,000 pfu.

An amount of 300,000 pfu (of amplified library) of the packaged cDNA wasplated at 20,000 pfu per 15 cm diameter plate after transfectingXL1-Blue cells. The plates were incubated at 37° C. for 8 h, then storedovernight at 4° C. Duplicate lifts were taken onto Colony/Plaque Screen™filters (DuPont) and treated as recommended by the manufacturer.

Preparation of PCR Templates

1. Plasmid DNA

DNA was isolated using an alkaline lysis procedure (Sambrook et al.;1989). Plasmid DNA was further purified by banding on a CsCl gradient.This DNA was used as template for PCR.

2. Chrysanthemum Genomic DNA

For isolation of total DNA, 5 g of Chrysanthemum petal tissue was frozenin liquid nitrogen and ground to a fine powder in a cold mortar andpestle. Ground tissue was extracted in 5 mL of phenol:chloroform,followed by 5 mL of NTMES buffer (0.01M NaCl; 0.1M Tris pH 8.5; 5 mMMgCl₂ ; 1 mM EDTA; 1% SDS). The aqueous phase was re-extracted with 5 mLof phenol:chloroform and the aqueous phase collected aftercentrifugation. DNA was spooled from this solution after addition of 0.5mL 3M NaAc, pH 5.8 and two volumes of ethanol. The final pellet wasresuspended in 2 mL TE buffer and the concentration determined prior touse in PCR.

3. Dianthus cDNA

Total RNA was isolated from the petal tissue of D. caryophyllus cv.Laguna stage 3 flowers, likewise using the method of Turpen and Griffith(1986). Poly(A)⁺ RNA was selected from the total RNA by Oligotex dT-30(Takana, Japan) following the manufacturer's protocol, and twomicrograms were reverse transcribed using Superscript™ reversetranscriptase as recommended by the manufacturer. The cDNA was dissolvedin 10 mL TE buffer. For PCR reactions, 5 mL were used as template.Conditions for PCR are described below.

4. Rosa cDNA

Total RNA was prepared from the buds of Rosa hybrida cv. Kardinalstage 1. At this stage, buds were 1.0-1.2 cm high and approximately 0.5cm wide. They were completely closed and no pigment was visible when thesepals were dissected away.

Frozen tissue (1-3 g) was ground in liquid nitrogen with a mortar andpestle, placed in 25 mL pre-chilled Buffer A [0.2M boric acid, 10 mMEDTA (sodium salt) (pH 7.6)] and homogenized briefly. The extract wasmixed on a rotary shaker until it reached room temperature and an equalvolume of phenol/chloroform (1:1 v/v), equilibrated with Buffer A, wasadded. After mixing for a further 10 min, the RNA preparation wascentrifuged at 10,000×g for 10 min at 20° C. The upper aqueous phase wasretained and the phenol interface re-extracted as above. The aqueousphases were pooled and adjusted to 0.1M sodium acetate (pH 6.0), 2.5volumes 95% ethanol were added and the mixture was stored at -20° C.overnight.

The preparation was centrifuged at 10,000×g for 10 min at 4° C., thepellet dissolved gently in 20 mL Buffer B [25 mM boric acid, 1.25 mMEDTA (sodium salt), 0.1M NaCl (pH 7.6)] and 0.4 volumes 2-butoxyethanol(2BE) were added. This solution was incubated on ice for 30 min. It wasthen centrifuged at 10,000×g for 10 min at 0° C. and the supernatantcarefully collected. After addition of 1.0 volume of 2BE and incubationon ice for a further 30 min, the supernatant was again centrifuged at10,000×g for 10 min at 0° C. The resulting pellet was gently washed withBuffer A:2BE (1:1 v/v), then with 70% (v/v) ethanol, 0.1M potassiumacetate and finally with 95% ethanol. The pellet was air dried anddissolved in 1 mL diethyl pyrocarbonate (DEPC)-treated water. This wasadjusted to 3M lithium chloride, left on ice for 60 min and centrifugedat 10,000×g for 10 min at 0° C. The pellet was washed twice with 3M LiCland then with 70% ethanol, 0.1M potassium acetate.

The resulting RNA pellet was dissolved in 400 mL DEPC-treated water andextracted with an equal volume phenol/chloroform. The RNA mix was thencentrifuged at 10,000×g for 5 min at 20° C., the aqueous phase collectedand made to 0.1M sodium acetate, and a further 2.5 volumes of 95%ethanol were added. After 30 min incubation on ice, the mix wascentrifuged at 13,000 rpm (5,000×g ) for 20 min at 20° C. and the RNApellet resuspended gently in 400 mL DEPC-treated water.

Poly (A)⁺ RNA was selected from the total RNA by Oligotex dT-30 (Takana,Japan) following the manufacturer's protocol.

Double-stranded cDNA was synthesized from 2 mg poly(A)⁺ RNA using thesame method as described above for the construction of the Petunia cDNAlibrary #2. The cDNA was dissolved in 10 mL TE buffer.

Synthesis of Oligonucleotides

Oligonucleotides and primers were synthesized on an Applied BiosystemsPCR-Mate DNA synthesizer using methods recommended by the manufacturer.The oligonucleotides and primers synthesized were, 5'-3':

    __________________________________________________________________________    Oligo 1 (SEQ ID NO:1):                                                                      GTTCAATTCGGAATGATG                                              Oligo 2 (SEQ ID NO:2):                                                                      GCTGCACTTAATCCATAT                                              Oligo 3 (SEQ ID NO:3):                                                                      GGATGACTCAAACAGCTATGACCATG                                      Oligo 4 (SEQ ID NO:4):                                                                      TGCATAGCTTTTGGG                                                 Oligo 5 (SEQ ID NO:5):                                                                      CCIGG(A/G)CAIATIC(G/T)                                                        (C/T)(C/T)TICCIGCICC                                                          (A/G)AAIGG                                                      Oligo 6 (SEQ ID NO:6):                                                                      GAGAGAGAGAGAGAGAGAG                                                           ATCTCGAGTTTTTTTTTTTTTTTTTT                                      Oligo 7 (SEQ ID NO:7):                                                                      CCIGC(A/G)CAIATIC(G/T)IC(T/G)                                                 ICCIGCICC(A/G)AAIGG                                             primer -40 (SEQ ID NO:8)                                                                    GTTTTCCCAGTCACGAC                                               primer 190 (SEQ ID NO:9)                                                                    TTGGAGTGGGCAATGGC                                               primer 191 (SEQ ID NO:10                                                                    CTGCTGCAAACAAGTCC                                               poly-T (SEQ ID NO:11)                                                                       TTTTTTTTTTTTTTTTT(AGC)                                          __________________________________________________________________________

The basis for the design of oligo 5 was as follows: Amino acid sequencesfrom the putative haem-binding domain of an avocado cytochrome P450(Bozak et al., 1990) and the corresponding sequences encoded by the twopetunia cytochrome P450 homologues pCGP142 and pCGP147 were aligned:

avocado(SEQ ID NO:12) P F G A G R R G C P G

pCGP142(SEQ ID NO:13) P F G A G K R I C P G

pCGP147(SEQ ID NO:14) P F G S G R R I C P G

The consensus amino acid sequence of the haem-binding region for thethree plant cytochromes P450 could thereby be seen to be:

(SEQ ID NO:15) P F G A(S) G R(K) R I(G) C P G

Possible permutations of nucleotide sequence that could encode the aminoacids found in the haem-binding domain of the three cytochrome P450molecules could then be deduced:

    ______________________________________                                        (SEQ ID NO:16)                                                                 ##STR1##                                                                     ______________________________________                                    

X indicates nucleotide positions where all four nucleotides (A,C,G andT) can be used. Oligo 5 was designed to complement a subset of theconsensus sequence derived from the three plant cytochromes P450.Deoxyinosine (I) was used predominantly when base degeneracy was greaterthan three. The resulting oligonucleotide sequence was as shown above.

Polymerase Chain Reactions

1. Amplification of Cloned Cytochrome P450 Sequences

For amplification of cloned Petunia cytochrome P450 sequences, PCR mixescontained 100 ng of plasmid template, 10 mM Tris-HCl (pH 8.3), 50 mMKCl, 1.25 mM MgCl₂, 0.2 mM each dNTP, 1.0 μM each primer and 0.5 unitAmpliTaq DNA Polymerase (Cetus). Reaction mixes (100 μl) were cycled 30times between 95° C. for 1 min, 42° C. for 1 min and 72° C. for 2 min.

2. Amplification of Dianthus Sequences Related to Petunia 3'-hydroxylase

PCR mixes contained 100 ng of cDNA template, 10 mM Tris-HCl (pH 8.3), 50mM KCl, 1.5 mM MgCl₂, 0.001% (w/v) gelatin, 0.2 mM each dNTP, 1.0 μMeach primer and 5 units AmpliTaq DNA Polymerase (Cetus). Reaction mixes(100 ml) were cycled firstly through 95° C. for 3 min, 55° C. for 1 minand 72° C. for 1 min, then through a further 39 cycles between 95° C.,55° C. and 72° C. each for 1 min. Amplified products were gel-purifiedusing Seaplaque low melting agarose (FMC). The mixture was heated untilthe agarose melted and extracted with TE-saturated phenol. The aqueousphase was then extracted with phenol/chloroform and the amplifiedproducts precipated with ethanol. Following gel-purification, theamplified products were cloned directly into the ddT-tailed pBluescriptvector described by Holton and Graham (1991).

3. Amplification of Chrysanthemum Sequences Related to Petunia3'-hydroxylase

Chrysanthemum reaction mixes contained 200 ng of genomic DNA template,10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl₂, 0.001% (w/v) gelatin,0.2 mM each dNTP, 1.0 μM each primer and 5 units AmpliTaq DNA Polymerase(Cetus). Reaction volumes of 50 mL were cycled 35 times between 95° C.,55° C. and 72° C., each for 90 s. Amplified products were gel-purifiedusing Geneclean (Bio 101 Inc.) and cloned directly into the ddT-tailedpBluescript vector described by Holton and Graham (1991).

4. Amplification of Rosa Sequences Related to Petunia 3'-hydroxylase

Rosa reaction mixes contained 1 μL of a 10-fold dilution of ds cDNAprepared as described above, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mMMgCl₂, 0.001% (w/v) gelatin, 0.2 mM each dNTP, 1.0 μM each primer and 5units AmpliTaq DNA Polymerase (Cetus). Reaction volumes of 50 mL werecycled 30 times between 95° C. for 1 min, 55° C. for 1 min and 72° C.for 3 min. Amplified products were gel-purified using Geneclean (Bio 101Inc.) and cloned directly into the ddT-tailed pBluescript vectordescribed by Holton and Graham (1991).

Screening of cDNA Libraries

Duplicate plaque lifts from cDNA library #2 were hybridized and washedas follows: High stringency conditions (hybridization: 50% (v/v)formamide, 6×SSC, 1% (w/v) SDS at 42° C. for 16 h and washing: 2×SSC, 1%SDS at 65° C. for 2×15 min followed by 0.2×SSC, 1% SDS at 65° C. for2×15 min) were used to detect sibling clones and low stringencyconditions (hybridization: 20% formamide, 6×SSC, 1% SDS at 42° C. for 16hand washing: 6×SSC, 1% SDS at 65° C. for 1 h) were used to detectrelated sequences.

Lifts from cDNA library #3 were hybridized and washed as follows: Forthe primary screening, using the Petunia 3'-hydroxylase cDNA EcoRI-XhoIinsert from pCGP619 (see FIG. 6), hybridization conditions were 20%(v/v) formamide, 1M NaCl, 10% (w/v) dextransulphate at 37° C. for 16 hand washing conditions were 0.1×SSC, 1% (w/v) SDS at room temperature.For the secondary screening, using the EcoRI-XhoI insert from pCGP854,conditions were identical except that the hybridization reaction tookplace at 42° C. for 16 h.

³² P-Labelling of DNA Probes

DNA fragments (50 to 100 ng) were radioactively labelled with 50 μCi of[α-³² P]-dCTP using an oligolabelling kit (Bresatec). Unincorporated[α-³² P]-dCTP was removed by chromatography on a Sephadex G-50 (Fine)column.

DNA Sequence Analysis

DNA sequencing was performed essentially by the method of Sanger et al.(1977) using the Sequenase enzyme (USB, version 2.1). The completesequence of clones pCGP602 and pCGP619 was determined by compilation ofsequence from different M13 -mp18 and -mp19 (Norrander et al., 1983;Yanisch-Perron, 1985) subclones obtained using standard cloningprocedures (Sambrook et al., 1989). For some regions it was necessary tosynthesise specific oligonucleotide primers to obtain overlappingsequence data, including primers -40, 190 191 and poly-T.

A restriction map of pCGP619 showing the position of several of thesesequences may be seen in FIG. 6.

Homology searches against Genbank, SWISS-PROT and EMBL databases wereperformed using the FASTA and TFASTA programs (Pearson and Lipman,1988).

3'-Hydroxylase Assay

3'-Hydroxylase enzyme activity was measured using a modified version ofthe method described by Stotz and Forkmann (1982). The assay reactionmixture typically contained 100 μL of yeast extract, 5 μL of 50 mM NADPHin assay buffer (100 mM potassium phosphate (pH 8.0), 1 mM EDTA and 20mM 2-mercaptoethanol) and 10 μCi of [³ H]-naringenin and was made up toa final volume of 210 μL with the assay buffer. Following incubation at23° C. for 2-16 h, the reaction mixture was extracted with 0.5 mL ofethylacetate. The ethylacetate phase was dried under vacuum and thenresuspended in 10 μL of ethylacetate. The tritiated flavonoid moleculeswere separated on cellulose thin layer plates (Merck Art 5577, Germany)using a chloroform: acetic acid: water (10:9:1 v/v) solvent system. Atthe completion of the chromatography the TLC plates were sprayed with 7%2,5-diphenyloxazol in diethyl ether. The reaction products werelocalised by autoradiography and identified by comparison tonon-radioactive naringenin and eriodictyol standards which were runalongside the reaction products and visualised under UV light.

Construction of pCGP621

A 1.8 kb EcoRI-XhoI fragment that included the entire cDNA insert frompCGP619 was ligated with the 8 kb EcoRI-SalI fragment from pYHCC101(Tanaka et al., 1988). The resulting plasmid, pCGP621, contained thepCGP619 cDNA fragment ligated in a sense orientation behind the yeastglyceraldehyde-3-phosphate dehydrogenase promoter.

Yeast Transformation

The yeast strain G-1315 (Mat α, trpl) (Ashikari et al., 1989) wastransformed with pCGP621 according to Ito et al. (1983). Thetransformants were selected by their ability to restore G-1315 totryptophan prototrophy.

Preparation of Yeast Extracts for Assay of 3'-hydroxylase Activity

A single isolate of G-1315/pCGP621 was used to inoculate 20 ml of YNBC[1.2% (w/v) yeast nitrogen base without amino acids (Difco), 2% (w/v)glucose and 0.3% (w/v) casamino acid (Difco)] which was subsequentlyincubated for 2 days at 30° C. Cells were collected by centrifugation,washed once with TE buffer, once with buffer A [10 mM Tris-HCl (pH 7.5),0.65M sorbitol, 0.1 mM DTT, 0.1 mM EDTA], and then resuspended in bufferB [10 mM Tris-HCl, (pH 7.5), 1.2M sorbitol, 0.1 mM DTT, 0.1 mM EDTA]containing zymolyase (0.1 mg/mL) (Seikagakukogyo, Japan). Followingincubation for 1 h at 30° C. the cells were pelleted by centrifugationand resuspended in 400 μL of buffer A. The cell suspension was thenvortexed with glass beads (diameter=0.4 mm) for 2 min and a 100 μLsample was assayed for activity.

Construction of pCGP293

The expression binary vector pCGP293 was derived from the Ti binaryvector pCGN1559 (McBride and Summerfelt, 1990). Plasmid pCGN1559 wasdigested with KpnI and the overhanging 3' ends were removed with T4 DNApolymerase according to standard protocols (Sambrook et al., 1989). Thevector was then further digested with XbaI and the resulting 5' overhangwas repaired using the Klenow fragment of DNA polymerase I. The vectorwas then re-ligated to give pCGP67. A 1.97 kb PstI fragment containingthe Mac promoter, mas terminator and various cloning sites (Comai etal., 1990) was isolated from pCGP40 and inserted into the Pstl site ofpCGP67 to give pCGP293.

Plasmid pCGP40 was constructed by removing the GUS gene (Jefferson etal., 1987) as a BamHI-SacI fragment from pCGN7334 and replacing it withthe BamHl-SacI fragment from pBluescribe M13⁻ that includes themulticloning site. Plasmid pCGN7334 (obtained from Calgene Inc., Calif.,USA), was constructed by inserting the fragment containing theMac-GUS-mas gene fusion into the XhoI site of pCGN7329 (Comai et al.,1990).

Construction of pCGP809

Plasmid pCGP809 was constructed by cloning the cDNA insert from pCGP619in a sense orientation behind the Mac promoter (Comai et al., 1990) ofpCGP293. The 1.8 kb BamHI-KpnI fragment containing the cDNA insert wasisolated from pCGP619 and ligated with a BamHI-KpnI digest of pCGP293.Correct insertion of the insert in pCGP809 was established byrestriction analysis of DNA isolated from gentamycin-resistanttransformants.

Petunia Transformation

a. Plant Material

Petunia hybrida (Skr4×Sw63) seeds were sterilized in 1.25% (w/v) sodiumhypochlorite for 10 minutes and rinsed three times in sterile water.Sterilized seeds were soaked in 100 mg/L gibberellic acid (GA₃) solutionfor 16 to 20 h. They were then germinated for 2 weeks on 10% (w/v) MS(Murashige and Skoog, 1962) medium supplemented with 1% (v/v) sucroseand 0.8% (w/v) Difco Bacto agar. Young seedlings were transferred to MSmedium supplemented with 3% (w/v) sucrose for 3 weeks before beingtransferred to Jiffy peat pellets (Jiffy Products Ltd, Norway), keptunder mist and illuminated (135 μE. mercury halide light, 22° C.) for 2to 3 weeks. These young plants were then transferred to a growth cabinet(68 μE. cool white fluorescent light, 25° C.). For co-cultivation, youngleaves were harvested and sterilized in 1.35% (w/v) sodium hypochloritefor 2 min followed by rinsing three times in sterile water. Leaf tissuewas then cut into 25 mm² squares and precultured on MS mediasupplemented with 0.05 mg/L kinetin and 1.0 mg/L2,4-dichlorophenoxyacetic acid (2,4-D) for 24 h.

b. Co-cultivation of Agrobacterium and Petunia Tissue

Agrobacterium tumefaciens strain AGL0 (Lazo et al., 1991) containing thebinary vector pCGP809 was maintained at 4° C. on MG/L (Garfinkel andNester, 1980) agar plates with 100 mg/L gentamycin. A single colony wasgrown overnight in liquid medium containing 1% (w/v) Bacto-peptone, 0.5%(w/v) Bacto-yeast extract and 1% (w/v) NaCl. A final concentration of5×10⁸ cells/mL was prepared the next day by dilution in liquid MS mediumcontaining 3% (w/v) sucrose (BPM). Leaf discs were dipped for 5 min intoBPM containing AGL0/pCGP809. The leaf discs were then blotted dry andplaced on co-cultivation media for 4 days. The co-cultivation mediumconsisted of SH medium (Schenk and Hilderbrandt, 1972) supplemented with0.05 mg/L kinetin and 1.0 mg/L 2,4-D and included a feeder layer oftobacco cell suspension spread over the co-cultivation medium with afilter paper placed on top of the tobacco cell suspension.

c. Recovery of Transgenic Petunia Plants

After co-cultivation, the leaf discs were transferred to selection mediaconsisting of fresh MS medium supplemented with 3% (w/v) sucrose, 2 mg/La-benzylaminopurine (BAP), 100 mg/L kanamycin, 350 mg/L cefotaxime, 0.3%(w/v) Gelrite Gellan Gum (Schweizerhall). After 3 weeks, regeneratingexplants were transferred to fresh medium. Adventitious shoots whichsurvived the kanamycin selection were isolated and transferred to BPMcontaining 100 mg/L kanamycin and 350 mg/L cefotaxime for rootinduction. All cultures were maintained under a 16 h photoperiod (60 μE.cool white fluorescent light) at 23±2° C. When roots reached 2-3 cm inlength the transgenic petunia plantlets were transferred to autoclavedDebco 51410/2 potting mix in 8 cm tubes. After 4 weeks plants werereplanted into 15 cm pots using the same potting mix and maintained at23° C. under a 14 h photoperiod (300 μE. mercury halide light).

2. Results

Isolation of Cytochrome P450 Homologues From cDNA Library #1

The isolation of five petunia cDNA clones that have regions of sequencesimilarity with cytochrome P450 enzymes has been described previously(International Patent Application No. PCT/AU92/00334). Partial sequencesof these clones, designated pCGP142, pCGP147, pCGP158, pCGP160 andpCGP454, are shown in FIG. 2. A mixed probe of ³² P-labelled DNAfragments that included the coding regions of these five cytochrome P450homologues (see FIGS. 2A and B) was used to screen 50,000 recombinantsfrom cDNA library #1 for related sequences. A total of 152 hybridizingclones were detected under low stringency hybridization and washingconditions. A further 13 different cytochrome P450 homologues wereidentified by sequence analysis of DNA isolated from the hybridizingclones.

One of these clones, designated pCGP174, was shown to correspond to theHf1 locus of Petunia (see International Patent Application No.PCT/AU92/00334). The nucleotide sequence of a full-length version ofthis clone, pCGP602, isolated from cDNA library #2 is shown in FIG. 3.Ten of the thirteen other cytochrome P450 homologues isolated in thescreen, pCGP161, pCGP162, pCGP163, pCGP165, pCGP166, pCGP167, pCGP168,pCGP169, pCGP171 and pCGP173 were used as a mixed probe to screen cDNAlibrary #2 for further cytochrome P450 homologues (see next section).

Isolation of the Cytochrome P450 Homologue pCGP619 from Petunia

A mixed probe of ³² P-labelled cDNA inserts from pCGP161, pCGP162,pCGP163, pCGP165, pCGP166, pCGP167, pCGP168, pCGP169, pCGP171 andpCGP173 (FIG. 4) was used to screen 1.5×10⁵ recombinants from cDNAlibrary #2. Over 200 hybridizing clones were detected with lowstringency hybridization and washing in 2×SSC and 1% SDS, at 65° C.Twenty-five of these clones hybridized to probes that included theinternal HincII-EcoRV and EcoRV-HindIII fragments of pCGP602 (FIG. 3)under low stringency conditions, but not under high stringencyconditions. Sequence analysis of this group of clones revealed thatseventeen were siblings of pCGP602 (shown previously to correspond tothe Hf1 locus of petunia--International Patent Application No.PCT/AU92/00334) and six were siblings of another petunia cDNA cloneencoded by the Hf2 locus (International Patent Application No.PCT/AU92/00334). One clone showed no sequence homology to cytochromesP450, and one, designated pCGP619, showed 57% and 39% sequence homologyto pCGP602 at the nucleotide and amino acid levels, respectively. Thecomplete nucleotide sequence and deduced amino acid sequence of thepCGP619 cDNA are shown in FIG. 5, and the restriction map outlining thesequencing strategy is shown in FIG. 6.

Expression of pCGP619 cDNA in Yeast

The cDNA insert from pCGP619 was ligated in a sense orientation behindthe glyceraldehyde-3-phosphate dehydrogenase promoter in the yeastvector pYHCC101. The resulting construct, designated pCGP621, was thentransformed into the yeast strain G-1315 (Ashikari et al., 1989).3'-Hydroxylase activity was detected in extracts of G-1315/pCGP621, butnot in extracts of the non-transgenic yeast (FIG. 7). From this it wasconcluded that the cDNA insert from pCGP619 encoded a 3'-hydroxylase.

Expression of a 3'-hydroxylase cDNA in Petunia

The binary plasmid construct designated pCGP809 was introduced into theF₁ petunia hybrid Skr4×Sw63 using Agrobacterium-mediated gene transfer.Leaf discs of Skr4×Sw63 were co-cultivated with AGL0/pCGP809 andintegration of the pCGP619 cDNA insert in the Skr4×Sw63 genome wasconfirmed by Southern analysis of plants obtained after kanamycinselection.

The expression of the introduced 3'-hydroxylase cDNA in the Skr4×Sw63hybrid had a noticeable effect on flower colour. In parts of the petalsof Skr4×Sw63 the colour changed from light pink to red. The colourchange observed may be described in terms of the numbers from the RoyalHorticultural Society's Colour Chart as having shifted from 55D-56C/D to54A-55A. Other biochemical and physiological conditions will affect theindividual outcome and the citing of the specific colour change achievedby expression of the 3'-hydroxylase cDNA in transgenic plants should notbe interpreted as limiting the possible range of colour changes whichmay be observed.

Generation of Mutants and Derivatives of Flavonoid 3'-hydroxylase

Using standard mutagenic techniques as hereinbefore disclosed, a rangeof mutants, derivatives and parts of flavonoid 3'-hydroxylase areobtainable, which may be useful in accordance with the presentinvention. For specific descriptions and protocols for such mutagenictechniques reference can conveniently be made to Sambrook et al. (1989).Examples of mutants, derivatives and parts of 3'-hydroxylase which areisolatable and contemplated herein include the following:

    __________________________________________________________________________    5' GCT                                                                              AAA                                                                              GAG                                                                              TTT                                                                              AAG                                                                              GAA                                                                              3'               (SEQ ID NO:17)                          Ala                                                                              Lys                                                                              Glu                                                                              Phe                                                                              Lys                                                                              Glu                    (SEQ ID NO:18)                          5' AAG                                                                              AAA                                                                              CTT                                                                              CCA                                                                              CCA                                                                              GGT                                                                              CCA                                                                              TTT                                                                              3'         (SEQ ID NO:19)                             Lys                                                                              Lys                                                                              Leu                                                                              Pro                                                                              Pro                                                                              Gly                                                                              Pro                                                                              Phe           (SEQ ID NO:20)                          5' TTG                                                                              GAG                                                                              TGG                                                                              GCA                                                                              ATG                                                                              GC 3'               (SEQ ID NO:21)                             Leu                                                                              Glu                                                                              Trp                                                                              Ala                                                                              Met                                                                              Ala                 (SEQ ID NO:22)                          5' G  GAC                                                                              TTG                                                                              TTT                                                                              GCA                                                                              GCA                                                                              GG 3'            (SEQ ID NO:23)                                Asp                                                                              Leu                                                                              Phe                                                                              Ala                                                                              Ala                                                                              Gly              (SEQ ID NO:24)                          5' CCA                                                                              TTT                                                                              GGT                                                                              GCT                                                                              GGT                                                                              CGA                                                                              AGA                                                                              ATT                                                                              TGC                                                                              CCT                                                                              GG 3'                                                                              (SEQ ID NO:25)                             Pro                                                                              Phe                                                                              Gly                                                                              Ala                                                                              Gly                                                                              Arg                                                                              Arg                                                                              Ile                                                                              Cys                                                                              Pro                                                                              Gly  (SEQ ID NO:26)                          __________________________________________________________________________

Detection of Related Sequences in Plant Species Other Than Petunia

Using standard Southern analysis techniques, a "nursery blot" wasprepared of DNA isolated from a variety of plant species, includingapple, carnation, cornflower, morning glory and rose to screen forgenetic sequences related to the petunia 3'-hydroxylase. Results clearlyshowed the presence of related genetic sequences in all the plantstested. The nursery blot comprised lanes 1-5 containing approximately 10mg DNA from each of the above-mentioned species, respectively. The probeDNA used was the HindIII-EcoRV fragment from pCGP619. Southern analysiswas conducted over a range of stringency conditions. Suitable stringencyconditions indicating the presence of a number of similar sequences ineach species were overnight incubation in 50% formamide, 1M NaCl, 1%SDS, 10% dextran sulphate at 42° C., followed by 3×30 min washes in2×SSC, 1% SDS at 60° C.

Isolation of a Cytochrome P450-homologous PCR Product From Rosa

Double-stranded rose petal cDNA, synthesized as described in Materialsand Methods, was used as the template for amplification of sequencesrelated to the petunia 3'-hydroxylase using oligonucleotides 7 and 190.A PCR product of approximately 400 bp was ligated into pBluescript andone of the recombinant plasmids recovered was designated pCGP635. Thenucleotide sequence and deduced amino acid sequence of the pCGP635insert are shown in FIG. 8. This insert shows 60% similarity at thenucleotide level to the Petunia pCGP619 cDNA.

Isolation of Cytochrome P450-homologous PCR Products From Dianthus

Single-stranded carnation petal cDNA synthesized as described inMaterials and Methods, was used as the template for amplification ofsequences related to the petunia 3'-hydroxylase using oligonucleotides 7and 190. A PCR product of approximately 400 bp was ligated intopBluescript. Sequence analysis of the recombinant plasmids revealed thattwo different cytochrome P450 homologues had been amplified and cloned.Representative clones of these two molecules were designated pCGP772 andpCGP773. The nucleotide sequence and deduced amino acid sequence of eachinsert are shown in FIGS. 9 and 10, respectively. Comparison of thededuced amino acid sequences with that of other cytochrome P450s yieldedthe following results:

    ______________________________________                                                         pCGP772  pCGP773                                             ______________________________________                                        pCGP619            59.2%      64.8%                                           pCGP158 (Haem binding area)                                                                      62.9%      61.1%                                           pCGP168 (Haem binding area)                                                                      59.5%                                                      Avocado cytochrome P450       57.8%                                           ______________________________________                                    

Isolation of a Cytochrome P450-homologous PCR Product From Chrysanthemum

Chrysanthemum genomic DNA isolated as described in the Materials andMethods was used as the template for amplification of sequences relatedto the petunia 3'-hydroxylase using oligonucleotides 7 and 190. A PCRproduct of approximately 400 bp was ligated into the ddT-tailedpBluescript and one of the recombinant plasmids recovered was designatedpCGP854. The nucleotide sequence and deduced amino acid sequence of 120of these base pairs are shown in FIG. 11. This sequence was comparedwith that from the Petunia cDNA clone pCGP619, shown in FIG. 5, andshows 73% and 65% similarity at the DNA and amino acid level,respectively, to the segment of sequence between positions 971 and 1091.

Isolation of a Chrysanthemum Petal cDNA Clone With Sequence Similarityto the Petunia 3'-hydroxylase

The cDNA insert from pCGP619 was used to screen cDNA library #3 forrelated sequences. Using the hybridization and washing conditionsdescribed in the Materials and Methods, 64 hybridizing clones weredetected. Twelve of these clones also hybridized to the insert frompCGP854. Sequence analysis of a putative full-length clone thathybridized to both the pCGP619 and pCGP854 probes revealed that itincluded an identical sequence to that of the PCR product sequence shownin FIG. 11 and therefore encodes a putative chrysanthemum3'-hydroxylase.

Expression of a Chrysanthemum Petal cDNA Clone in Yeast

The petal cDNA clone can be ligated in a sense orientation behind theglyceraldehyde-3-phosphate dehydrogenase promoter in the yeast vectorpYHCC101. The resulting construct is then transformed into the yeaststrain G-1315 (Ashikari et al., 1989). Activity of the 3'-hydroxylasecan be detected in extracts of G-1315 plus construct, but not inextracts of non-transgenic yeast. From this result it can be concludedthat the cDNA insert encodes a 3'-hydroxylase.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

REFERENCES

Ashikari, T., Kiuchi-Goto, N., Tanaka, Y., Shibano, Y., Amachi, T., andYoshizumi, H. Appl. Microbiol. Biotechnol. 30: 515-520, 1989.

Aviv, H. and Leder, P., Proc. Natl. Acad. Sci. USA 69: 1408-1412, 1972.

Bethesda Research Laboratories. BRL pUC host: E. coli DH5a™ competentcells. Bethesda Res. Lab. Focus. 8(2): 9, 1986.

Bozak, K. R., Yu, H., Sirevag, R. and Christoffersen, R. E., Proc. Natl.Acad. Sci. USA 87: 3904-3908, 1990.

Bullock, W. O., Fernandez, J. M. and Short, J. M. Biotechniques 5: 376,1987.

Comai, L., Moran, P. and Maslyar, D., Plant Molecular Biology 15:373-381, 1990.

Ebel, J. and Hahlbrock, K., In The Flavonoids: Advances in ResearchSince 1980. Harborne, J. B. (Ed.), Academic Press, New York, USA,641-679, 1988.

Forkmann, G. Plant Breeding 106: 1-26, 1991.

Garfinkel, D. J. and Nester, E. W. J. Bacteriol. 144: 732-743, 1980.

Hahlbrock, K. and Grisebach, H. Annu. Rev. Plant Physiol. 30: 105-130,1979.

Hanahan, D. J. Mol. Biol. 166: 557, 1983.

Holton, T. A. and Graham, M. W. Nucleic Acids Research 19: 1156, 1991.

Inoue, H., Nojima, H. and Okayama, H. Gene 96: 23-28, 1990.

Ito, H., Fukuda, Y., Murata, K. and Kimura, A. J. Bacteriol., 153:163-168, 1983.

Jefferson, R. A., Kavanagh, T. A. and Bevan, M. W. EMBO J. 6(13):3901-3907, 1987.

Lazo, G. R., Pascal, A. S. and Ludwig, R. A. Bio/technology 9: 963-967,1991.

McBride, K. E. and Sumerfelt, K. R. Plant Molecular Biology 14: 269-2761990.

Merrifield, J. Am. Chem. Soc. 85: 2149, 1964.

Murashige, T. and Skoog, F. Physiol. Plant 15: 73-97, 1962.

Norrander, J., Kemp, T. and Messing, J. Gene 26: 101, 1983.

Pearson, W. R. and Lipman, D. J. Proc. Natl. Acad. Sci. USA 85:2444-2448, 1988.

Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: ALaboratory Manual (2nd edition). Cold Spring Harbor Laboratory Press,USA, 1989.

Sanger, F., Nicklen, S. and Coulson, A. Proc. Natl. Acad. Sci. USA 74:5463-5467, 1977.

Schenk, R. U. and Hilderbrandt, A. C. Can. J. Bot. 50: 199-204, 1972.

Schram, A. W., Jonsson, L. M. V. and Bennink, G. J. H. Biochemistry offlavonoid synthesis in Petunia hybrida. In: Petunia Sink, K. C. (Ed.)Springer-Verlag, Berlin, Germany pp 68-75, 1984.

Stafford, H. A. Flavonoid Metabolism. CRC Press, Inc. Boca Raton, Fla.,USA, 1990.

Stotz, G. and Forkmann, G. Z. Naturforsch 37c: 19-23, 1982.

Tanaka, Y., Ashikari, T., Shibano, Y., Amachi, T., Yoshizumi, H. andMatsubara, H. J. Biochem. 103: 954-961, 1988.

Turpen, T. H. and Griffith, O. M. BioTechniques 4: 11-15, 1986.

Wiering, H. and De Vlaming, P. Inheritance and Biochemistry of Pigments.In: Petunia Sink, K. C. (Ed.), Springer-Verlag, Berlin, Germany pp49-65, 1984.

Yanisch-Perron, C., Vieira, J. and Messing, J. Gene 33: 103, 1985.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 58                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GTTCAATTCGGAATGATG18                                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GCTGCACTTAATCCATAT18                                                          (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GGATGACTCAAACAGCTATGACCATG26                                                  (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       TGCATAGCTTTTGGG15                                                             (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 3..4                                                            (D) OTHER INFORMATION: /mod.sub.-- base=i                                     (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 9..10                                                           (D) OTHER INFORMATION: /mod.sub.-- base=i                                     (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 12..13                                                          (D) OTHER INFORMATION: /mod.sub.-- base=i                                     (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 18..19                                                          (D) OTHER INFORMATION: /mod.sub.-- base=i                                     (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 21..22                                                          (D) OTHER INFORMATION: /mod.sub.-- base=i                                     (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 24..25                                                          (D) OTHER INFORMATION: /mod.sub.-- base=i                                     (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 30..31                                                          (D) OTHER INFORMATION: /mod.sub.-- base=i                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CCNGGRCANATNCKYYTNCCNGCNCCRAANGG32                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GAGAGAGAGAGAGAGAGAGATCTCGAGTTTTTTTTTTTTTTTTTT45                               (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 3..4                                                            (D) OTHER INFORMATION: /mod.sub.-- base=i                                     (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 9..10                                                           (D) OTHER INFORMATION: /mod.sub.-- base=i                                     (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 12..13                                                          (D) OTHER INFORMATION: /mod.sub.-- base=i                                     (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 15..16                                                          (D) OTHER INFORMATION: /mod.sub.-- base=i                                     (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 18..19                                                          (D) OTHER INFORMATION: /mod.sub.-- base=i                                     (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 21..22                                                          (D) OTHER INFORMATION: /mod.sub.-- base=i                                     (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 24..25                                                          (D) OTHER INFORMATION: /mod.sub.-- base=i                                     (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 30..31                                                          (D) OTHER INFORMATION: /mod.sub.-- base=i                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CCNGCRCANATNCKNCKNCCNGCNCCRAANGG32                                            (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GTTTTCCCAGTCACGAC17                                                           (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       TTGGAGTGGGCAATGGC17                                                           (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      CTGCTGCAAACAAGTCC17                                                           (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      TTTTTTTTTTTTTTTTTAGC20                                                        (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      ProPheGlyAlaGlyArgArgGlyCysProGly                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      ProPheGlyAlaGlyLysArgIleCysProGly                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      ProPheGlySerGlyArgArgIleCysProGly                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      ProPheGlyAlaSerGlyArgLysArgIleGlyCysProGly                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      CCNTTTGGNGCNGGNAGNCGNATNTGTCCNGGN33                                           (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..18                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      GCTAAAGAGTTTAAGGAA18                                                          AlaLysGluPheLysGlu                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      AlaLysGluPheLysGlu                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..24                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      AAGAAACTTCCACCAGGTCCATTT24                                                    LysLysLeuProProGlyProPhe                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      LysLysLeuProProGlyProPhe                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..17                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      TTGGAGTGGGCAATGGC17                                                           LeuGluTrpAlaMet                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      LeuGluTrpAlaMet                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 2..18                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      GGACTTGTTTGCAGCAGG18                                                          AspLeuPheAlaAla                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      AspLeuPheAlaAla                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..32                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      CCATTTGGTGCTGGTCGAAGAATTTGCCCTGG32                                            ProPheGlyAlaGlyArgArgIleCysPro                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      ProPheGlyAlaGlyArgArgIleCysPro                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 733 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..402                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      TTTAGTTCAATTCGGAATGATGAGATTTCGAGTCTCATTTCATCAATT48                            PheSerSerIleArgAsnAspGluIleSerSerLeuIleSerSerIle                              151015                                                                        CATTCCATGAACGGTTCTGTTGTCAACATGACACAAAAGATTCTTTGT96                            HisSerMetAsnGlySerValValAsnMetThrGlnLysIleLeuCys                              202530                                                                        TTTACAAACTCTGTGACTTGTAGAACAGCTTTCGGGAAAGTATACAAA144                           PheThrAsnSerValThrCysArgThrAlaPheGlyLysValTyrLys                              354045                                                                        AATCAAAATGAATTGATAAACTTGATGAGGGAAGTACTGGAATTAGTA192                           AsnGlnAsnGluLeuIleAsnLeuMetArgGluValLeuGluLeuVal                              505560                                                                        GGAGGATTTGATTTTGAAAATTCTCCGGTTGAGTTTATTGGAAATCAC240                           GlyGlyPheAspPheGluAsnSerProValGluPheIleGlyAsnHis                              65707580                                                                      TTTGAGCTTGTTCCGTTTGGTGCAGGAAAAAGGATTTGTCCAGGAATG288                           PheGluLeuValProPheGlyAlaGlyLysArgIleCysProGlyMet                              859095                                                                        CAATTTGGTTTAGCTAATATTAGACATCCTTTGGCTCGATTCCTCTAC336                           GlnPheGlyLeuAlaAsnIleArgHisProLeuAlaArgPheLeuTyr                              100105110                                                                     CATTTTAACTGGGCGCTTCCATATGAAACTAATCCTGAAGATTTAGAT384                           HisPheAsnTrpAlaLeuProTyrGluThrAsnProGluAspLeuAsp                              115120125                                                                     AGTCTGAAAAATATGGATTAAGTGCAGCAAAAGAGAAAGATCTATACT432                           SerLeuLysAsnMetAsp                                                            130                                                                           TAATTGCCGTAGATCACAAAGAAGGTGATATATAAATTCTGATGTTCTGCTTTAAATGGT492               GAAAGTCATACTCTACACAATGCTTCATCTCCTTAATTTGAGTTTGGTGTACATTTGTGT552               CTCCCTTTTAGCTTTGAATTTCACCTTGAAAAATGATCACATTTTCTTTTTCTGTTACTC612               CAATTAAGATATATGTTGTGGTTGGTCAATTATGCCATATTTATCAAAAGATCAAATCAA672               TTCCCTCGTTGATAAGTATAGATTATAAAACTGATTAATGAATCAAAAAAAAAAAAAAAA732               A733                                                                          (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 134 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      PheSerSerIleArgAsnAspGluIleSerSerLeuIleSerSerIle                              151015                                                                        HisSerMetAsnGlySerValValAsnMetThrGlnLysIleLeuCys                              202530                                                                        PheThrAsnSerValThrCysArgThrAlaPheGlyLysValTyrLys                              354045                                                                        AsnGlnAsnGluLeuIleAsnLeuMetArgGluValLeuGluLeuVal                              505560                                                                        GlyGlyPheAspPheGluAsnSerProValGluPheIleGlyAsnHis                              65707580                                                                      PheGluLeuValProPheGlyAlaGlyLysArgIleCysProGlyMet                              859095                                                                        GlnPheGlyLeuAlaAsnIleArgHisProLeuAlaArgPheLeuTyr                              100105110                                                                     HisPheAsnTrpAlaLeuProTyrGluThrAsnProGluAspLeuAsp                              115120125                                                                     SerLeuLysAsnMetAsp                                                            130                                                                           (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1665 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 3..1432                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      TGCAATTTTTCAACTTGGTTTCCTTTCTCCTTATTGTATTTTCCCTC47                             GlnPhePheAsnLeuValSerPheLeuLeuIleValPheSerLeu                                 151015                                                                        ATTTCATTAAGAAAATGGAAGAAATCCAATTGTCAAACCAAAAAATTG95                            IleSerLeuArgLysTrpLysLysSerAsnCysGlnThrLysLysLeu                              202530                                                                        CCTCCAGGCCCATGGAAAGTACCTTTTCTTGGAAGCTTGCTTCATATG143                           ProProGlyProTrpLysValProPheLeuGlySerLeuLeuHisMet                              354045                                                                        GTAGGTGGACTTCCACACCATGTCCTTAGAGATTTAGCCAAAAAATAT191                           ValGlyGlyLeuProHisHisValLeuArgAspLeuAlaLysLysTyr                              505560                                                                        GGACCAATTATGCACCTTCAACTAGGTAAAATTTCTGCCGTTGTAGTT239                           GlyProIleMetHisLeuGlnLeuGlyLysIleSerAlaValValVal                              657075                                                                        ACTTCTCCTGAGATGGCAAGAAAAGTACTAAAAACTCATGACCTTGCA287                           ThrSerProGluMetAlaArgLysValLeuLysThrHisAspLeuAla                              80859095                                                                      TTTGCATATAGGCCTAAACTTCTAGGCATTGAGATTGTCTGCTATAAT335                           PheAlaTyrArgProLysLeuLeuGlyIleGluIleValCysTyrAsn                              100105110                                                                     AGTTCAGACATTGCCTTTTCCCCGTATGGTGATTACTGGAGGCAAATG383                           SerSerAspIleAlaPheSerProTyrGlyAspTyrTrpArgGlnMet                              115120125                                                                     CGTAAAATTTGTGTATTGGAAGTGCTTAGTGCCAAAAATGTCCGGTCA431                           ArgLysIleCysValLeuGluValLeuSerAlaLysAsnValArgSer                              130135140                                                                     TTTAACTCGATTAGACGAGATGAAATACTTCTTATGATCGATTTTTTG479                           PheAsnSerIleArgArgAspGluIleLeuLeuMetIleAspPheLeu                              145150155                                                                     CGATCATCTTCTGGTAAGCCAGTTAATATAACAGAAAGGATCTTTTCA527                           ArgSerSerSerGlyLysProValAsnIleThrGluArgIlePheSer                              160165170175                                                                  TTCACAAGCTCTATGATTTGTAGATCAGTATTTGGGAAAAGAATAAAG575                           PheThrSerSerMetIleCysArgSerValPheGlyLysArgIleLys                              180185190                                                                     GAGAAAGACGAATGTATACGACATGTGAAAAAAATGACAGGCTTAATA623                           GluLysAspGluCysIleArgHisValLysLysMetThrGlyLeuIle                              195200205                                                                     GATGGGTTCGATGTGGCTGACATATTCCCTTCGTTGAGGTTTCTTCAT671                           AspGlyPheAspValAlaAspIlePheProSerLeuArgPheLeuHis                              210215220                                                                     GTACTAATCGGTATGAAGGGTAAAATTATGGATGTTCATCGTAAGGTA719                           ValLeuIleGlyMetLysGlyLysIleMetAspValHisArgLysVal                              225230235                                                                     GATGCTATTGTTGAGGAAGTCATGAATGAGCACAAAGAAACTCTTCGA767                           AspAlaIleValGluGluValMetAsnGluHisLysGluThrLeuArg                              240245250255                                                                  ACTGGCAAGACCAATGGTGAAGTGGGAGGAGAAGATTTAATTGATGTA815                           ThrGlyLysThrAsnGlyGluValGlyGlyGluAspLeuIleAspVal                              260265270                                                                     TTGCTAAGACTTAAGGAAGAGGGAGACCTTCAACTTCCAATCACAAAT863                           LeuLeuArgLeuLysGluGluGlyAspLeuGlnLeuProIleThrAsn                              275280285                                                                     GACAACATCAAAGCCATTTTTAATGACATGTTTGCTGCGGGAACAGAA911                           AspAsnIleLysAlaIlePheAsnAspMetPheAlaAlaGlyThrGlu                              290295300                                                                     ACTTCATCAACAACAATTAACTGGGCCATGGTAGAACTGATGAAAAAT959                           ThrSerSerThrThrIleAsnTrpAlaMetValGluLeuMetLysAsn                              305310315                                                                     CCAAGTGTATTCGCGAAAGCTCAAGCAGAGGTAAGAGAAGTCTTCAAA1007                          ProSerValPheAlaLysAlaGlnAlaGluValArgGluValPheLys                              320325330335                                                                  GGGAAAGAAACTTTCGATGAAGATGATATCGAGGAGCTGAATTACCTT1055                          GlyLysGluThrPheAspGluAspAspIleGluGluLeuAsnTyrLeu                              340345350                                                                     AAGTTAGTCATTAGAGAAACTTTAAGACTCCACCCTCCACTTCCACTT1103                          LysLeuValIleArgGluThrLeuArgLeuHisProProLeuProLeu                              355360365                                                                     TTGCTTCCAAGAGAATGTCGGAGAGAAACAGAAATAAATGGCTACACT1151                          LeuLeuProArgGluCysArgArgGluThrGluIleAsnGlyTyrThr                              370375380                                                                     ATTCCTTTAAATACCAAAGTCATAGTTAATGTTTGGGCTATTGGAAGA1199                          IleProLeuAsnThrLysValIleValAsnValTrpAlaIleGlyArg                              385390395                                                                     GATCCAAAATATTGGGATGATGCAGAAAGCTTTAAGCCTGAGAGATTT1247                          AspProLysTyrTrpAspAspAlaGluSerPheLysProGluArgPhe                              400405410415                                                                  GAACATAACTCTTTGAATTTTGCTGGCAATAATTTTGAATATCTTCCT1295                          GluHisAsnSerLeuAsnPheAlaGlyAsnAsnPheGluTyrLeuPro                              420425430                                                                     TTTGGTAGTGGAAGGAGGATTTGCCCCGGAATATCATTTGGTTTAGCT1343                          PheGlySerGlyArgArgIleCysProGlyIleSerPheGlyLeuAla                              435440445                                                                     AATGTTTATCATCCATTGGCTCAATTGTTGTATCATTTCGATTGGAGA1391                          AsnValTyrHisProLeuAlaGlnLeuLeuTyrHisPheAspTrpArg                              450455460                                                                     CTTCCTACTGGGGTCGACCCAAATGACTTTGAATTGACTAGTTAGCTGGAG1442                       LeuProThrGlyValAspProAsnAspPheGluLeuThr                                       465470475                                                                     TAACTACTGGTAGGAAAAGAGACCTTTACTTGATTTTCACTCCTTATTCACCTTCTCTAA1502              AGTGATTAAATGGGCAAATTTTAATTTGAAATAATACTTTTTCTTGTTTACATTTCTCTC1562              CCATTGTTGTATTTCATTTACCTATTGTTGTACTTCTTTCTTTTGTTGATGTCTTAGGTT1622              TTACCTATTTCTATGCATTTGTATTTAAAAAAAAAAAAAAAAA1665                               (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 476 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      GlnPhePheAsnLeuValSerPheLeuLeuIleValPheSerLeuIle                              151015                                                                        SerLeuArgLysTrpLysLysSerAsnCysGlnThrLysLysLeuPro                              202530                                                                        ProGlyProTrpLysValProPheLeuGlySerLeuLeuHisMetVal                              354045                                                                        GlyGlyLeuProHisHisValLeuArgAspLeuAlaLysLysTyrGly                              505560                                                                        ProIleMetHisLeuGlnLeuGlyLysIleSerAlaValValValThr                              65707580                                                                      SerProGluMetAlaArgLysValLeuLysThrHisAspLeuAlaPhe                              859095                                                                        AlaTyrArgProLysLeuLeuGlyIleGluIleValCysTyrAsnSer                              100105110                                                                     SerAspIleAlaPheSerProTyrGlyAspTyrTrpArgGlnMetArg                              115120125                                                                     LysIleCysValLeuGluValLeuSerAlaLysAsnValArgSerPhe                              130135140                                                                     AsnSerIleArgArgAspGluIleLeuLeuMetIleAspPheLeuArg                              145150155160                                                                  SerSerSerGlyLysProValAsnIleThrGluArgIlePheSerPhe                              165170175                                                                     ThrSerSerMetIleCysArgSerValPheGlyLysArgIleLysGlu                              180185190                                                                     LysAspGluCysIleArgHisValLysLysMetThrGlyLeuIleAsp                              195200205                                                                     GlyPheAspValAlaAspIlePheProSerLeuArgPheLeuHisVal                              210215220                                                                     LeuIleGlyMetLysGlyLysIleMetAspValHisArgLysValAsp                              225230235240                                                                  AlaIleValGluGluValMetAsnGluHisLysGluThrLeuArgThr                              245250255                                                                     GlyLysThrAsnGlyGluValGlyGlyGluAspLeuIleAspValLeu                              260265270                                                                     LeuArgLeuLysGluGluGlyAspLeuGlnLeuProIleThrAsnAsp                              275280285                                                                     AsnIleLysAlaIlePheAsnAspMetPheAlaAlaGlyThrGluThr                              290295300                                                                     SerSerThrThrIleAsnTrpAlaMetValGluLeuMetLysAsnPro                              305310315320                                                                  SerValPheAlaLysAlaGlnAlaGluValArgGluValPheLysGly                              325330335                                                                     LysGluThrPheAspGluAspAspIleGluGluLeuAsnTyrLeuLys                              340345350                                                                     LeuValIleArgGluThrLeuArgLeuHisProProLeuProLeuLeu                              355360365                                                                     LeuProArgGluCysArgArgGluThrGluIleAsnGlyTyrThrIle                              370375380                                                                     ProLeuAsnThrLysValIleValAsnValTrpAlaIleGlyArgAsp                              385390395400                                                                  ProLysTyrTrpAspAspAlaGluSerPheLysProGluArgPheGlu                              405410415                                                                     HisAsnSerLeuAsnPheAlaGlyAsnAsnPheGluTyrLeuProPhe                              420425430                                                                     GlySerGlyArgArgIleCysProGlyIleSerPheGlyLeuAlaAsn                              435440445                                                                     ValTyrHisProLeuAlaGlnLeuLeuTyrHisPheAspTrpArgLeu                              450455460                                                                     ProThrGlyValAspProAsnAspPheGluLeuThr                                          465470475                                                                     (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 547 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..514                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      GGGATGATGAAGCAAGGAGATTTCTTGGATGTACTTCTTGATCAATGT48                            GlyMetMetLysGlnGlyAspPheLeuAspValLeuLeuAspGlnCys                              151015                                                                        GATGAAGAAGGGTCTGGATTTGATCGCCAAACTATCAAGCCTCTCATC96                            AspGluGluGlySerGlyPheAspArgGlnThrIleLysProLeuIle                              202530                                                                        CTGGATTTATTCATTGCTGGAAGTGATACATCTGCCATAACAACAGAA144                           LeuAspLeuPheIleAlaGlySerAspThrSerAlaIleThrThrGlu                              354045                                                                        TGGGCAATGGCAGAACTACTTCGAAAACCTCAAGAATTTGTGAATGCA192                           TrpAlaMetAlaGluLeuLeuArgLysProGlnGluPheValAsnAla                              505560                                                                        TGGGCAATTGGAAGAGATCCAAAATACTGGGAAAAACCACTGGAGTTT240                           TrpAlaIleGlyArgAspProLysTyrTrpGluLysProLeuGluPhe                              65707580                                                                      ATGCCTGAAAGATTCTTGAAGTGTAGTTTGGATTACAAAGGTAGGGNN288                           MetProGluArgPheLeuLysCysSerLeuAspTyrLysGlyArgXaa                              859095                                                                        TTTGAGTATATACCATTTGGCGCAGGTCGAAGAATTTGTCCTGGAATG336                           PheGluTyrIleProPheGlyAlaGlyArgArgIleCysProGlyMet                              100105110                                                                     CCACATTGCAATAAGGATGGTGAATTTGATGCTGGCTTCGATTATTCA384                           ProHisCysAsnLysAspGlyGluPheAspAlaGlyPheAspTyrSer                              115120125                                                                     CCATTTAGTTGGGAATTACCTNAAGGAATGGCACCAAAGNATTTGAAC432                           ProPheSerTrpGluLeuProXaaGlyMetAlaProLysXaaLeuAsn                              130135140                                                                     ATGGAGGAACAGTTTGGAGTTACCTTGAGGAAGGCTATTCCCCTTATT480                           MetGluGluGlnPheGlyValThrLeuArgLysAlaIleProLeuIle                              145150155160                                                                  GCCATTCCCAGTATGGAAGAAAAGGTCATATTTTAGCCCAAAAG524                               AlaIleProSerMetGluGluLysValIlePhe                                             165170                                                                        CTATGCATTTTGTGTGTATGTTT547                                                    (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 171 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      GlyMetMetLysGlnGlyAspPheLeuAspValLeuLeuAspGlnCys                              151015                                                                        AspGluGluGlySerGlyPheAspArgGlnThrIleLysProLeuIle                              202530                                                                        LeuAspLeuPheIleAlaGlySerAspThrSerAlaIleThrThrGlu                              354045                                                                        TrpAlaMetAlaGluLeuLeuArgLysProGlnGluPheValAsnAla                              505560                                                                        TrpAlaIleGlyArgAspProLysTyrTrpGluLysProLeuGluPhe                              65707580                                                                      MetProGluArgPheLeuLysCysSerLeuAspTyrLysGlyArgXaa                              859095                                                                        PheGluTyrIleProPheGlyAlaGlyArgArgIleCysProGlyMet                              100105110                                                                     ProHisCysAsnLysAspGlyGluPheAspAlaGlyPheAspTyrSer                              115120125                                                                     ProPheSerTrpGluLeuProXaaGlyMetAlaProLysXaaLeuAsn                              130135140                                                                     MetGluGluGlnPheGlyValThrLeuArgLysAlaIleProLeuIle                              145150155160                                                                  AlaIleProSerMetGluGluLysValIlePhe                                             165170                                                                        (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 618 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..336                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      AAACAGATCAATGCATTGCTTGTGGAAATATTTGGAGCTGGTACAGAA48                            LysGlnIleAsnAlaLeuLeuValGluIlePheGlyAlaGlyThrGlu                              151015                                                                        TCTACAACTGCTACAAGCCAATGGATGCTTGTAGAACTCCTTAGAAAT96                            SerThrThrAlaThrSerGlnTrpMetLeuValGluLeuLeuArgAsn                              202530                                                                        CGACAAGCCTTGCCCAAAGACACTCAAGTTATGGTAAACGAGTGGGCG144                           ArgGlnAlaLeuProLysAspThrGlnValMetValAsnGluTrpAla                              354045                                                                        ATTGCGTATGATCCTAAGATTTGGGGCAGCTTCAAACCCGAAAGGTTT192                           IleAlaTyrAspProLysIleTrpGlySerPheLysProGluArgPhe                              505560                                                                        ATCGATTCAAAAATAGATCCTTTGGACCACAAAGGGCAAAATTTTGAA240                           IleAspSerLysIleAspProLeuAspHisLysGlyGlnAsnPheGlu                              65707580                                                                      TATTTTCCTTTTGGTTCTGGAAGGAGAATTTGTGCTGGAGAACCTTTG288                           TyrPheProPheGlySerGlyArgArgIleCysAlaGlyGluProLeu                              859095                                                                        GCTTCTAGGGTTATTCCCTTAGCTGTTGCTTCTATGATCCATAAGTTT336                           AlaSerArgValIleProLeuAlaValAlaSerMetIleHisLysPhe                              100105110                                                                     GATATCACTATGTTAGAAGATCCACTCTCATCATTCCTAAGTTGAGAAGAGTGAGGAAAT396               TAAAAGAAGCAGAAGATATGTTACTATAAAAACTCGTTATATATATATATATTGCTGTAT456               CTATATATGTGTGAATGATCTGCTGCTCATGTTGTGTTTTGTTGTTTGTGTACTATAGGT516               CATACCTAAGTTGATGAAATGTCTCTGAGAATATATACTCCTTATATAATAGGAGTAATT576               TACCGATAATTAATATTCCTGCGACAAAAAAAAAAAAAAAAA618                                 (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 112 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      LysGlnIleAsnAlaLeuLeuValGluIlePheGlyAlaGlyThrGlu                              151015                                                                        SerThrThrAlaThrSerGlnTrpMetLeuValGluLeuLeuArgAsn                              202530                                                                        ArgGlnAlaLeuProLysAspThrGlnValMetValAsnGluTrpAla                              354045                                                                        IleAlaTyrAspProLysIleTrpGlySerPheLysProGluArgPhe                              505560                                                                        IleAspSerLysIleAspProLeuAspHisLysGlyGlnAsnPheGlu                              65707580                                                                      TyrPheProPheGlySerGlyArgArgIleCysAlaGlyGluProLeu                              859095                                                                        AlaSerArgValIleProLeuAlaValAlaSerMetIleHisLysPhe                              100105110                                                                     (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 203 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 3..203                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      CTCGAGAATCAATGGAAGATGTAAGATTACTAGGCTATCACATACCT47                             ArgGluSerMetGluAspValArgLeuLeuGlyTyrHisIlePro                                 151015                                                                        GCTAAAACGAGACTCTTTATCAATGCTTGGACAATGGGGAGAGACCCA95                            AlaLysThrArgLeuPheIleAsnAlaTrpThrMetGlyArgAspPro                              202530                                                                        CTAACATGGGAAAATCCAGAAGAGTATCAGCCAGAGAGATTCTTGAAT143                           LeuThrTrpGluAsnProGluGluTyrGlnProGluArgPheLeuAsn                              354045                                                                        AGAGATACTGATGTCAAAGGAGTAAACTTTGAGTTCATTCCCTTTGGC191                           ArgAspThrAspValLysGlyValAsnPheGluPheIleProPheGly                              505560                                                                        GCCGGCAGAAGC203                                                               AlaGlyArgSer                                                                  65                                                                            (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 67 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      ArgGluSerMetGluAspValArgLeuLeuGlyTyrHisIleProAla                              151015                                                                        LysThrArgLeuPheIleAsnAlaTrpThrMetGlyArgAspProLeu                              202530                                                                        ThrTrpGluAsnProGluGluTyrGlnProGluArgPheLeuAsnArg                              354045                                                                        AspThrAspValLysGlyValAsnPheGluPheIleProPheGlyAla                              505560                                                                        GlyArgSer                                                                     65                                                                            (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1812 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 126..1643                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      CTTTCTACTAGCTACTTCGTTATATATATGTAAAATTGTGACTTTGAAAATCATTTAAAT60                TATCATAAGGTTCATTTTATCTTGATCAAAATATTTACTTCGGCCATATACGTTTTCCTT120               TAGTCATGATGCTACTTACTGAGCTTGGTGCAGCAACTTCAATCTTT167                            MetMetLeuLeuThrGluLeuGlyAlaAlaThrSerIlePhe                                    1510                                                                          CTAATAGCACACATAATCATTTCAACTCTTATTTCAAAAACTACCGGC215                           LeuIleAlaHisIleIleIleSerThrLeuIleSerLysThrThrGly                              15202530                                                                      CGGCATCTACCGCCGGGGCCAAGAGGGTGGCCGGTGATCGGAGCACTT263                           ArgHisLeuProProGlyProArgGlyTrpProValIleGlyAlaLeu                              354045                                                                        CCACTTTTAGGAGCCATGCCACATGTTTCCTTAGCTAAAATGGCAAAA311                           ProLeuLeuGlyAlaMetProHisValSerLeuAlaLysMetAlaLys                              505560                                                                        AAATATGGAGCAATCATGTATCTCAAAGTTGGAACATGTGGCATGGCA359                           LysTyrGlyAlaIleMetTyrLeuLysValGlyThrCysGlyMetAla                              657075                                                                        GTTGCTTCTACCCCTGATGCTGCTAAAGCATTCTTGAAAACACTTGAT407                           ValAlaSerThrProAspAlaAlaLysAlaPheLeuLysThrLeuAsp                              808590                                                                        ATCAACTTCTCCAATCGTCCACCTAATGCAGGTGCCACTCACTTAGCT455                           IleAsnPheSerAsnArgProProAsnAlaGlyAlaThrHisLeuAla                              95100105110                                                                   TATAATGCTCAAGACATGGTTTTTGCACATTATGGACCACGATGGAAG503                           TyrAsnAlaGlnAspMetValPheAlaHisTyrGlyProArgTrpLys                              115120125                                                                     TTGCTAAGGAAATTAAGCAACTTGCATATGCTAGGGGGAAAAGCCTTA551                           LeuLeuArgLysLeuSerAsnLeuHisMetLeuGlyGlyLysAlaLeu                              130135140                                                                     GAGAATTGGGCAAATGTTCGTGCCAATGAGCTAGGGCACATGCTAAAA599                           GluAsnTrpAlaAsnValArgAlaAsnGluLeuGlyHisMetLeuLys                              145150155                                                                     TCAATGTCCGATATGAGTCGAGAGGGCCAGAGGGTTGTGGTGGCGGAG647                           SerMetSerAspMetSerArgGluGlyGlnArgValValValAlaGlu                              160165170                                                                     ATGTTGACATTTGCCATGGCCAATATGATCGGACAAGTGATGCTAAGC695                           MetLeuThrPheAlaMetAlaAsnMetIleGlyGlnValMetLeuSer                              175180185190                                                                  AAAAGAGTATTTGTAGATAAAGGTGTTGAGGTAAATGAATTTAAGGAC743                           LysArgValPheValAspLysGlyValGluValAsnGluPheLysAsp                              195200205                                                                     ATGGTTGTAGAGTTAATGACAATAGCAGGGTATTTCAACATTGGTGAT791                           MetValValGluLeuMetThrIleAlaGlyTyrPheAsnIleGlyAsp                              210215220                                                                     TTTATTCCTTGTTTAGCTTGGATGGATTTACAAGGGATAGAAAAACGA839                           PheIleProCysLeuAlaTrpMetAspLeuGlnGlyIleGluLysArg                              225230235                                                                     ATGAAACGTTTACATAAGAAGTTTGATGCTTTATTGACAAAGATGTTT887                           MetLysArgLeuHisLysLysPheAspAlaLeuLeuThrLysMetPhe                              240245250                                                                     GATGAACACAAAGCAACTACCTATGAACGTAAGGGGAAACCAGATTTT935                           AspGluHisLysAlaThrThrTyrGluArgLysGlyLysProAspPhe                              255260265270                                                                  CTTGATGTTGTTATGGAAAATGGGGACAATTCTGAAGGAGAAAGACTC983                           LeuAspValValMetGluAsnGlyAspAsnSerGluGlyGluArgLeu                              275280285                                                                     AGTACAACCAACATCAAAGCACTTTTGCTGAATTTGTTCACAGCTGGT1031                          SerThrThrAsnIleLysAlaLeuLeuLeuAsnLeuPheThrAlaGly                              290295300                                                                     ACGGACACTTCTTCTAGTGCAATAGAATGGGCACTTGCAGAAATGATG1079                          ThrAspThrSerSerSerAlaIleGluTrpAlaLeuAlaGluMetMet                              305310315                                                                     AAGAACCCTGCCATTTTGAAAAAAGCACAAGCAGAAATGGATCAAGTC1127                          LysAsnProAlaIleLeuLysLysAlaGlnAlaGluMetAspGlnVal                              320325330                                                                     ATTGGAAGAAATAGGCGTTTACTCGAATCCGATATCCCAAATCTCCCT1175                          IleGlyArgAsnArgArgLeuLeuGluSerAspIleProAsnLeuPro                              335340345350                                                                  TACCTCCGAGCAATTTGCAAAGAAACATTTCGAAAACACCCTTCTACA1223                          TyrLeuArgAlaIleCysLysGluThrPheArgLysHisProSerThr                              355360365                                                                     CCATTAAATCTTCCTAGGATCTCGAACGAACCATGCATAGTCGATGGT1271                          ProLeuAsnLeuProArgIleSerAsnGluProCysIleValAspGly                              370375380                                                                     TATTACATACCAAAAAACACTAGGCTTAGTGTTAACATATGGGCAATT1319                          TyrTyrIleProLysAsnThrArgLeuSerValAsnIleTrpAlaIle                              385390395                                                                     GGAAGAGATCCCCAAGTTTGGGAAAATCCACTAGAGTTTAATCCCGAA1367                          GlyArgAspProGlnValTrpGluAsnProLeuGluPheAsnProGlu                              400405410                                                                     AGATTCTTGAGTGGAAGAAACTCCAAGATTGATCCTCGAGGGAACGAT1415                          ArgPheLeuSerGlyArgAsnSerLysIleAspProArgGlyAsnAsp                              415420425430                                                                  TTTGAATTGATACCATTTGGTGCTGGACGAAGAATTTGTGCAGGAACA1463                          PheGluLeuIleProPheGlyAlaGlyArgArgIleCysAlaGlyThr                              435440445                                                                     AGAATGGGAATTGTAATGGTGGAATATATATTAGGAACTTTGGTTCAT1511                          ArgMetGlyIleValMetValGluTyrIleLeuGlyThrLeuValHis                              450455460                                                                     TCATTTGATTGGAAATTACCAAGTGAAGTTATTGAGTTGAATATGGAA1559                          SerPheAspTrpLysLeuProSerGluValIleGluLeuAsnMetGlu                              465470475                                                                     GAAGCTTTTGGCTTAGCTTTGCAGAAAGCTGTCCCTCTTGAAGCTATG1607                          GluAlaPheGlyLeuAlaLeuGlnLysAlaValProLeuGluAlaMet                              480485490                                                                     GTTACTCCAAGGTTACAATTGGATGTTTATGTACCATAGCTATAGA1653                            ValThrProArgLeuGlnLeuAspValTyrValPro                                          495500505                                                                     TGTGTATTGTGCTATAATTGCGCATGTTGTTGGTTGTAGCATGAGATATTAAAAGGAGTA1713              CATGAAGCGCATTGCATGAGTTTAACTTGTAGCTCCTTAATATTTTAGGTATTTTTCAAT1773              TAATAAGTTCTTGTTGGTTGGGTAAAAAAAAAAAAAAAA1812                                   (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 506 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      MetMetLeuLeuThrGluLeuGlyAlaAlaThrSerIlePheLeuIle                              151015                                                                        AlaHisIleIleIleSerThrLeuIleSerLysThrThrGlyArgHis                              202530                                                                        LeuProProGlyProArgGlyTrpProValIleGlyAlaLeuProLeu                              354045                                                                        LeuGlyAlaMetProHisValSerLeuAlaLysMetAlaLysLysTyr                              505560                                                                        GlyAlaIleMetTyrLeuLysValGlyThrCysGlyMetAlaValAla                              65707580                                                                      SerThrProAspAlaAlaLysAlaPheLeuLysThrLeuAspIleAsn                              859095                                                                        PheSerAsnArgProProAsnAlaGlyAlaThrHisLeuAlaTyrAsn                              100105110                                                                     AlaGlnAspMetValPheAlaHisTyrGlyProArgTrpLysLeuLeu                              115120125                                                                     ArgLysLeuSerAsnLeuHisMetLeuGlyGlyLysAlaLeuGluAsn                              130135140                                                                     TrpAlaAsnValArgAlaAsnGluLeuGlyHisMetLeuLysSerMet                              145150155160                                                                  SerAspMetSerArgGluGlyGlnArgValValValAlaGluMetLeu                              165170175                                                                     ThrPheAlaMetAlaAsnMetIleGlyGlnValMetLeuSerLysArg                              180185190                                                                     ValPheValAspLysGlyValGluValAsnGluPheLysAspMetVal                              195200205                                                                     ValGluLeuMetThrIleAlaGlyTyrPheAsnIleGlyAspPheIle                              210215220                                                                     ProCysLeuAlaTrpMetAspLeuGlnGlyIleGluLysArgMetLys                              225230235240                                                                  ArgLeuHisLysLysPheAspAlaLeuLeuThrLysMetPheAspGlu                              245250255                                                                     HisLysAlaThrThrTyrGluArgLysGlyLysProAspPheLeuAsp                              260265270                                                                     ValValMetGluAsnGlyAspAsnSerGluGlyGluArgLeuSerThr                              275280285                                                                     ThrAsnIleLysAlaLeuLeuLeuAsnLeuPheThrAlaGlyThrAsp                              290295300                                                                     ThrSerSerSerAlaIleGluTrpAlaLeuAlaGluMetMetLysAsn                              305310315320                                                                  ProAlaIleLeuLysLysAlaGlnAlaGluMetAspGlnValIleGly                              325330335                                                                     ArgAsnArgArgLeuLeuGluSerAspIleProAsnLeuProTyrLeu                              340345350                                                                     ArgAlaIleCysLysGluThrPheArgLysHisProSerThrProLeu                              355360365                                                                     AsnLeuProArgIleSerAsnGluProCysIleValAspGlyTyrTyr                              370375380                                                                     IleProLysAsnThrArgLeuSerValAsnIleTrpAlaIleGlyArg                              385390395400                                                                  AspProGlnValTrpGluAsnProLeuGluPheAsnProGluArgPhe                              405410415                                                                     LeuSerGlyArgAsnSerLysIleAspProArgGlyAsnAspPheGlu                              420425430                                                                     LeuIleProPheGlyAlaGlyArgArgIleCysAlaGlyThrArgMet                              435440445                                                                     GlyIleValMetValGluTyrIleLeuGlyThrLeuValHisSerPhe                              450455460                                                                     AspTrpLysLeuProSerGluValIleGluLeuAsnMetGluGluAla                              465470475480                                                                  PheGlyLeuAlaLeuGlnLysAlaValProLeuGluAlaMetValThr                              485490495                                                                     ProArgLeuGlnLeuAspValTyrValPro                                                500505                                                                        (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 180 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      CCAGACACCCACAAACTTCCATACCTTCAGGCTGTGATCAAGGAGACTCTTCGTCTCCGG60                ATGGCAATTCCTCTATTAGTCCCACACATGAAACTTTACAGAAAACGTTCATCTTTTTAT120               GTCATATCAAGTCTTCTTGGACTGGTTCGTTATTACACCTACCTATCTGAATGTATTTTT180               (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 180 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      ACGAACATGGGAAAATCCAGAAGAGTATCAGCCAGAGAGATTCTTGAATAGTGATATTGA60                TGTCAAAGGACTAAACTTTGAGTTGATTCCATGGCTAGTAGCTACTTCTTTCATGATATC120               TGTAATAAGTGTAGTGCTCGACTCCTTCAGGCGAGTTGTGTGTTTAATTTCTCCAGTATC180               (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 180 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      AAGTTCTTTCCATCAGTTATCAAACAAACCATGAGGCTGCATCCCCCTCTCCCTTTATTA60                CTATTAAGGGAAAGCAAGGAATCTTGTGAAGATAGGGAGCGGTTTACTCCCTTCGTGGCC120               TTACCATTACACTAACAATGAATGGGCTTGGAAATAGTCTCAGATGTTTTTAAAGAAAAC180               (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 179 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      GTGATTTTCCAAAAGAATCTCACTTGCTGCAGATGTCCTATGTTCAAGCCTGTGTGAAGG60                AAACTCTTAGGTTGCATCCTCCGGCGCCATTATTTATTGAAGTTGAGAAACTTATGTATG120               AAAGTGTCATACAGAACTACTGCCCATGTGGTGTGTTTTAGTACTTCTTTTTTTTGGGT179                (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 180 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      ATGTCCTATGTTCAAGCCTGTGTGCCGGAAACTCTTAGGTTGCATCCTCCGGCGCCATTG60                CTACTTCCACATGCGTGCAATCGAAACATGTCCCCCTGGATTTGTACACTAGATACAAGA120               CTTAGCGGTCCTGGTGTAATCTCAATTCTCATGTGGTTATAAACAGAAGTTCTTCTGGTG180               (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 180 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      GAAACATTATCAATGAACATGTTAAGAATCGAGCACTCGGAAGCAAGGGAAATGGTGCGT60                TTGGAGGTGAAGATTTGGTTGATGTTTTACGGTTGGGGTAAATTGGGGCCCCCCTTTTAA120               GGCTTTGGAATTTCCACCTGGAAAAATGGACCCCATTTTCCTTTTCCTGTACCTCCAATT180               (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 90 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      AAAACTGCAAACTAGCGATAACGCTGATGTTCTTGATGTGTTGTTGCATACTAGCGAGGA60                AGATCCAGAGGCAATCGACAGAATTCACAT90                                              (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 180 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      CAGTACACTCTTTGTGTTCATCATATCTCTTCACATTGCTCACAAGCTCGACCATGGCCG60                TCGGTAAGAACAAGAGGATTTCCAAAGGCAAATACCACATTCTGATGATTCACTTGATAT120               ATGTGTACCTTTATGTCATTTAATGGCACAACAATTCTGGGGACTTAGGTTCAAAGAAGC180               (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 180 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      CTGTAGGGTTACCGTTCATTGGAAATTTGCATCAATATGATACTTTAAAGCCGCATATCT60                ACTTCTGGAAACTTTCTAGGAAGTATGGAATACTTTCGGTTTTGAATTATGTATACATAT120               ATAAAACAAATGTGAAATGTATACATATAATAAAATTGCTCTCATGATATACTTCTCTAT180               (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 179 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      TTCTGGAAATGTTTCTAGCTGGTACAGAGACATCTAGCAGCACAACAGAGTGGGCACTAA60                CTGAACTCCTTCGAAACCCAGAAACAATGGACAATTCTTACGCTGAATTTGTTGTTCGCC120               CTTTTATTTTCAGTTTGATTGTATCCAAAGGATGTCGAATGAAATCATACTCTTTACCT179                (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1757 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 35..1522                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      CCGTTGCTGTCGAGAAAACAGAAAGAAGAGAAAAATGGACTACGTGAATATT52                        MetAspTyrValAsnIle                                                            15                                                                            TTGCTGGGACTGTTTTTCACTTGGTTCTTGGTGAATGGACTCATGTCA100                           LeuLeuGlyLeuPhePheThrTrpPheLeuValAsnGlyLeuMetSer                              101520                                                                        CTTCGAAGAAGAAAAATCTCTAAGAAACTTCCACCAGGTCCATTTCCT148                           LeuArgArgArgLysIleSerLysLysLeuProProGlyProPhePro                              253035                                                                        TTGCCTATCATCGGAAATCTTCACTTACTTGGTAATCATCCTCACAAA196                           LeuProIleIleGlyAsnLeuHisLeuLeuGlyAsnHisProHisLys                              404550                                                                        TCACTTGCTCAACTTGCAAAAATTCATGGTCCTATTATGAATCTCAAA244                           SerLeuAlaGlnLeuAlaLysIleHisGlyProIleMetAsnLeuLys                              55606570                                                                      TTAGGCCAACTAAACACAGTGGTCATTTCATCATCAGTCGTGGCAAGA292                           LeuGlyGlnLeuAsnThrValValIleSerSerSerValValAlaArg                              758085                                                                        GAAGTCTTGCAAAAACAAGACTTAACATTTTCCAATAGGTTTGTCCCG340                           GluValLeuGlnLysGlnAspLeuThrPheSerAsnArgPheValPro                              9095100                                                                       GACGTAGTCCATGTCCGAAATCACTCCGATTTTTCTGTTGTTTGGTTA388                           AspValValHisValArgAsnHisSerAspPheSerValValTrpLeu                              105110115                                                                     CCAGTCAATTCTCGATGGAAAACGCTTCGCAAAATCATGAACTCTAGC436                           ProValAsnSerArgTrpLysThrLeuArgLysIleMetAsnSerSer                              120125130                                                                     ATCTTTTCTGGTAACAAGCTTGATGGTAATCAACATCTGAGGTCTAAA484                           IlePheSerGlyAsnLysLeuAspGlyAsnGlnHisLeuArgSerLys                              135140145150                                                                  AAGGTCCAAGAGTTAATTGATTATTGTCAAAAGTGTGCCAAGAATGGC532                           LysValGlnGluLeuIleAspTyrCysGlnLysCysAlaLysAsnGly                              155160165                                                                     GAAGCAGTGGATATAGGAAGAGCAACTTTTGGAACTACTTTGAATTTG580                           GluAlaValAspIleGlyArgAlaThrPheGlyThrThrLeuAsnLeu                              170175180                                                                     CTATCCAACACCATTTTCTCTAAAGATTTGACTAATCCGTTTTCTGAT628                           LeuSerAsnThrIlePheSerLysAspLeuThrAsnProPheSerAsp                              185190195                                                                     TCTGCTAAAGAGTTTAAGGAATTGGTTTGGAACATTATGGTTGAGGCT676                           SerAlaLysGluPheLysGluLeuValTrpAsnIleMetValGluAla                              200205210                                                                     GGAAAACCCAATTTGGTGGACTACTTTCCTTTCCTTGAGAAAATTGAT724                           GlyLysProAsnLeuValAspTyrPheProPheLeuGluLysIleAsp                              215220225230                                                                  CCGCAAGGTATAAAGCGACGCATGACTAATAATTTTACTAAGTTTCTT772                           ProGlnGlyIleLysArgArgMetThrAsnAsnPheThrLysPheLeu                              235240245                                                                     GGCCTTATCAGCGGTTTGATTGATGACCGGTTAAAGGAAAGGAATCTA820                           GlyLeuIleSerGlyLeuIleAspAspArgLeuLysGluArgAsnLeu                              250255260                                                                     AGGGACAATGCAAATATTGATGTTTTAGACGCCCTTCTCAACATTAGC868                           ArgAspAsnAlaAsnIleAspValLeuAspAlaLeuLeuAsnIleSer                              265270275                                                                     CAAGAGAACCCAGAAGAGATTGACAGGAATCAAATCGAGCAGTTGTGT916                           GlnGluAsnProGluGluIleAspArgAsnGlnIleGluGlnLeuCys                              280285290                                                                     CTGGACTTGTTTGCAGCAGGGACTGATACTACATCGAATACCTTGGAG964                           LeuAspLeuPheAlaAlaGlyThrAspThrThrSerAsnThrLeuGlu                              295300305310                                                                  TGGGCAATGGCAGAACTACTTCAGAATCCACACACATTGCAGAAAGCA1012                          TrpAlaMetAlaGluLeuLeuGlnAsnProHisThrLeuGlnLysAla                              315320325                                                                     CAAGAAGAACTTGCACAAGTCATTGGTAAAGGCAAACAAGTAGAAGAA1060                          GlnGluGluLeuAlaGlnValIleGlyLysGlyLysGlnValGluGlu                              330335340                                                                     GCAGATGTTGGACGACTACCTTACTTGCGATGCATAGTGAAAGAAACC1108                          AlaAspValGlyArgLeuProTyrLeuArgCysIleValLysGluThr                              345350355                                                                     TTACGAATACACCCAGCGGCTCCTCTCTTAATTCCACGTAAAGTGGAG1156                          LeuArgIleHisProAlaAlaProLeuLeuIleProArgLysValGlu                              360365370                                                                     GAAGACGTTGAGTTGTCTACCTATATTATTCCAAAGGATTCACAAGTT1204                          GluAspValGluLeuSerThrTyrIleIleProLysAspSerGlnVal                              375380385390                                                                  CTAGTGAACGTATGGGCAATTGGACGCAACTCTGATCTATGGGAAAAT1252                          LeuValAsnValTrpAlaIleGlyArgAsnSerAspLeuTrpGluAsn                              395400405                                                                     CCTTTGGTCTTTAAGCCAGAAAGGTTTTGGGAGTCAGAAATAGATATC1300                          ProLeuValPheLysProGluArgPheTrpGluSerGluIleAspIle                              410415420                                                                     CGAGGTCGAGATTTTGAACTCATTCCATTTGGTGCTGGTCGAAGAATT1348                          ArgGlyArgAspPheGluLeuIleProPheGlyAlaGlyArgArgIle                              425430435                                                                     TGCCCTGGATTGCCTTTGGCTATGAGGATGATTCCAGTAGCACTAGGT1396                          CysProGlyLeuProLeuAlaMetArgMetIleProValAlaLeuGly                              440445450                                                                     TCATTGCTAAACTCATTTAATTGGAAACTATATGGTGGAATTGCACCT1444                          SerLeuLeuAsnSerPheAsnTrpLysLeuTyrGlyGlyIleAlaPro                              455460465470                                                                  AAAGATTTGGACATGCAGGAAAAGTTTGGCATTACCTTGGCGAAAGCC1492                          LysAspLeuAspMetGlnGluLysPheGlyIleThrLeuAlaLysAla                              475480485                                                                     CAACCTCTGCTAGCTATCCCAACTCCCCTGTAGCTATAGGGATAAATTAA1542                        GlnProLeuLeuAlaIleProThrProLeu                                                490495                                                                        GTTGAGGTTTTAAGTTACTAGTAGATTCTATTGCAGCTATAGGATTTCTTTCACCATCAC1602              GTATGCTTTACCGTTGGATGATGGAAAGAAATATCTATAGCTTTGGGTTTGTTTAGTTTG1662              CACATAAAAATTGAATGAATGGAATACCATGGAGTTATAAGAAATAATAAGACTATGATT1722              CTTACCCTACTTGAACAATGACATGGCTATTTCAC1757                                       (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 496 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      MetAspTyrValAsnIleLeuLeuGlyLeuPhePheThrTrpPheLeu                              151015                                                                        ValAsnGlyLeuMetSerLeuArgArgArgLysIleSerLysLysLeu                              202530                                                                        ProProGlyProPheProLeuProIleIleGlyAsnLeuHisLeuLeu                              354045                                                                        GlyAsnHisProHisLysSerLeuAlaGlnLeuAlaLysIleHisGly                              505560                                                                        ProIleMetAsnLeuLysLeuGlyGlnLeuAsnThrValValIleSer                              65707580                                                                      SerSerValValAlaArgGluValLeuGlnLysGlnAspLeuThrPhe                              859095                                                                        SerAsnArgPheValProAspValValHisValArgAsnHisSerAsp                              100105110                                                                     PheSerValValTrpLeuProValAsnSerArgTrpLysThrLeuArg                              115120125                                                                     LysIleMetAsnSerSerIlePheSerGlyAsnLysLeuAspGlyAsn                              130135140                                                                     GlnHisLeuArgSerLysLysValGlnGluLeuIleAspTyrCysGln                              145150155160                                                                  LysCysAlaLysAsnGlyGluAlaValAspIleGlyArgAlaThrPhe                              165170175                                                                     GlyThrThrLeuAsnLeuLeuSerAsnThrIlePheSerLysAspLeu                              180185190                                                                     ThrAsnProPheSerAspSerAlaLysGluPheLysGluLeuValTrp                              195200205                                                                     AsnIleMetValGluAlaGlyLysProAsnLeuValAspTyrPhePro                              210215220                                                                     PheLeuGluLysIleAspProGlnGlyIleLysArgArgMetThrAsn                              225230235240                                                                  AsnPheThrLysPheLeuGlyLeuIleSerGlyLeuIleAspAspArg                              245250255                                                                     LeuLysGluArgAsnLeuArgAspAsnAlaAsnIleAspValLeuAsp                              260265270                                                                     AlaLeuLeuAsnIleSerGlnGluAsnProGluGluIleAspArgAsn                              275280285                                                                     GlnIleGluGlnLeuCysLeuAspLeuPheAlaAlaGlyThrAspThr                              290295300                                                                     ThrSerAsnThrLeuGluTrpAlaMetAlaGluLeuLeuGlnAsnPro                              305310315320                                                                  HisThrLeuGlnLysAlaGlnGluGluLeuAlaGlnValIleGlyLys                              325330335                                                                     GlyLysGlnValGluGluAlaAspValGlyArgLeuProTyrLeuArg                              340345350                                                                     CysIleValLysGluThrLeuArgIleHisProAlaAlaProLeuLeu                              355360365                                                                     IleProArgLysValGluGluAspValGluLeuSerThrTyrIleIle                              370375380                                                                     ProLysAspSerGlnValLeuValAsnValTrpAlaIleGlyArgAsn                              385390395400                                                                  SerAspLeuTrpGluAsnProLeuValPheLysProGluArgPheTrp                              405410415                                                                     GluSerGluIleAspIleArgGlyArgAspPheGluLeuIleProPhe                              420425430                                                                     GlyAlaGlyArgArgIleCysProGlyLeuProLeuAlaMetArgMet                              435440445                                                                     IleProValAlaLeuGlySerLeuLeuAsnSerPheAsnTrpLysLeu                              450455460                                                                     TyrGlyGlyIleAlaProLysAspLeuAspMetGlnGluLysPheGly                              465470475480                                                                  IleThrLeuAlaLysAlaGlnProLeuLeuAlaIleProThrProLeu                              485490495                                                                     (2) INFORMATION FOR SEQ ID NO:51:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 390 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 3..389                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                      TTTTGGAGTGGGCAATGGCCGAAATCTTGAGGCATCCCAGAGTTTGT47                             LeuGluTrpAlaMetAlaGluIleLeuArgHisProArgValCys                                 151015                                                                        AGAAAAATGCAAAATGAGGCGATGGAGATTGCTAATGGCAAACCACAC95                            ArgLysMetGlnAsnGluAlaMetGluIleAlaAsnGlyLysProHis                              202530                                                                        ATCACAGAAAGTGATTTAGATAAAATGCACTACTTGAAAGCAGTGATC143                           IleThrGluSerAspLeuAspLysMetHisTyrLeuLysAlaValIle                              354045                                                                        AAAGAGACACTTCGGCTACATCCGCCAATACCATTACTCTCCCCTCGT191                           LysGluThrLeuArgLeuHisProProIleProLeuLeuSerProArg                              505560                                                                        GAATCAACTGAAGATGTTAAGATAATGGAATCTGACATAGAAGTCAAA239                           GluSerThrGluAspValLysIleMetGluSerAspIleGluValLys                              657075                                                                        AAACTATGGTCTTTATCAATGCTTGGGCAATCGGAAGAGACCCAGCAG287                           LysLeuTrpSerLeuSerMetLeuGlyGlnSerGluGluThrGlnGln                              80859095                                                                      AGTGGGATGAACCAAGAGTTTCGACCGGAGAGATTCATGAATTCTTCT335                           SerGlyMetAsnGlnGluPheArgProGluArgPheMetAsnSerSer                              100105110                                                                     GTGGATTTCAAAGGTCATCTCTTTCAATTACTCCCCTTCGGAGCCGGC383                           ValAspPheLysGlyHisLeuPheGlnLeuLeuProPheGlyAlaGly                              115120125                                                                     CGCAGAT390                                                                    ArgArg                                                                        (2) INFORMATION FOR SEQ ID NO:52:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 129 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                      LeuGluTrpAlaMetAlaGluIleLeuArgHisProArgValCysArg                              151015                                                                        LysMetGlnAsnGluAlaMetGluIleAlaAsnGlyLysProHisIle                              202530                                                                        ThrGluSerAspLeuAspLysMetHisTyrLeuLysAlaValIleLys                              354045                                                                        GluThrLeuArgLeuHisProProIleProLeuLeuSerProArgGlu                              505560                                                                        SerThrGluAspValLysIleMetGluSerAspIleGluValLysLys                              65707580                                                                      LeuTrpSerLeuSerMetLeuGlyGlnSerGluGluThrGlnGlnSer                              859095                                                                        GlyMetAsnGlnGluPheArgProGluArgPheMetAsnSerSerVal                              100105110                                                                     AspPheLysGlyHisLeuPheGlnLeuLeuProPheGlyAlaGlyArg                              115120125                                                                     Arg                                                                           (2) INFORMATION FOR SEQ ID NO:53:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 377 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 3..377                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                      TGGCGGAACTACTGCGCAACCCCGAGAAAATGGCAAAAGCACAAGAC47                             AlaGluLeuLeuArgAsnProGluLysMetAlaLysAlaGlnAsp                                 151015                                                                        GAAATAGACCGAATAGTAGGCGACAAGAACAAATCGTTCCAAGAGACA95                            GluIleAspArgIleValGlyAspLysAsnLysSerPheGlnGluThr                              202530                                                                        GACATCTCAAAGTTACCGTACATTCAAGCGGTTGTTAAAGAAACATTA143                           AspIleSerLysLeuProTyrIleGlnAlaValValLysGluThrLeu                              354045                                                                        AGGCTACACCCGCCTGGACCGTTCCTAATACCCCACAAAGCCGAAAAG191                           ArgLeuHisProProGlyProPheLeuIleProHisLysAlaGluLys                              505560                                                                        GACGTAAACTTAAGCCGGTTTTTCATCCCCGAGGACGCCCAAGTGTGG239                           AspValAsnLeuSerArgPhePheIleProGluAspAlaGlnValTrp                              657075                                                                        GTCAATGTATGGGCCATTGGTCGTGATCCAAGCGTGTGGCGGGTCCCA287                           ValAsnValTrpAlaIleGlyArgAspProSerValTrpArgValPro                              80859095                                                                      CTTACATTGTGTCCTGAACGGTTTTTGGAAAACGACATCGATTTCAAA335                           LeuThrLeuCysProGluArgPheLeuGluAsnAspIleAspPheLys                              100105110                                                                     GGTACAGATTTCGAGCTGATTCCCTTTGGCGCCGGCCGCATC377                                 GlyThrAspPheGluLeuIleProPheGlyAlaGlyArgIle                                    115120125                                                                     (2) INFORMATION FOR SEQ ID NO:54:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 125 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                      AlaGluLeuLeuArgAsnProGluLysMetAlaLysAlaGlnAspGlu                              151015                                                                        IleAspArgIleValGlyAspLysAsnLysSerPheGlnGluThrAsp                              202530                                                                        IleSerLysLeuProTyrIleGlnAlaValValLysGluThrLeuArg                              354045                                                                        LeuHisProProGlyProPheLeuIleProHisLysAlaGluLysAsp                              505560                                                                        ValAsnLeuSerArgPhePheIleProGluAspAlaGlnValTrpVal                              65707580                                                                      AsnValTrpAlaIleGlyArgAspProSerValTrpArgValProLeu                              859095                                                                        ThrLeuCysProGluArgPheLeuGluAsnAspIleAspPheLysGly                              100105110                                                                     ThrAspPheGluLeuIleProPheGlyAlaGlyArgIle                                       115120125                                                                     (2) INFORMATION FOR SEQ ID NO:55:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 386 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 2..385                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                      AATGGCAGAGCTGCTCCGTAACCCAGAAAAACTGAAGAAAGCACAA46                              MetAlaGluLeuLeuArgAsnProGluLysLeuLysLysAlaGln                                 151015                                                                        GTAGAGCTTCAAGAAATCATCGGCAGAGGAAACACATTAGAGGAATCT94                            ValGluLeuGlnGluIleIleGlyArgGlyAsnThrLeuGluGluSer                              202530                                                                        GACATCAGTCGATTGCCATATTTACAGGCTATCATTAAGGAAACATTT142                           AspIleSerArgLeuProTyrLeuGlnAlaIleIleLysGluThrPhe                              354045                                                                        CGGCTACACCCAGGACTGCCATTATTGCTACCTAGGAAAGTTGGTTCA190                           ArgLeuHisProGlyLeuProLeuLeuLeuProArgLysValGlySer                              505560                                                                        GACGTTCAGCTCTTTGGGTTTACAGTACCCAAAAATGCACAAGTCATA238                           AspValGlnLeuPheGlyPheThrValProLysAsnAlaGlnValIle                              657075                                                                        ATCAACGCCTGGGCAATTGGGAGAGACCCAGATTGTTGGCAGAAACCC286                           IleAsnAlaTrpAlaIleGlyArgAspProAspCysTrpGlnLysPro                              80859095                                                                      AACTCATTTGAGCCAGAAAGGTTCCTTGGGTCACAAATTGATGTGAAG334                           AsnSerPheGluProGluArgPheLeuGlySerGlnIleAspValLys                              100105110                                                                     GGTCGTGATTTTGAGCTAATTCCCTTTGGCGCCGGCCGCAGCATCTGT382                           GlyArgAspPheGluLeuIleProPheGlyAlaGlyArgSerIleCys                              115120125                                                                     GCCG386                                                                       Ala                                                                           (2) INFORMATION FOR SEQ ID NO:56:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 128 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                      MetAlaGluLeuLeuArgAsnProGluLysLeuLysLysAlaGlnVal                              151015                                                                        GluLeuGlnGluIleIleGlyArgGlyAsnThrLeuGluGluSerAsp                              202530                                                                        IleSerArgLeuProTyrLeuGlnAlaIleIleLysGluThrPheArg                              354045                                                                        LeuHisProGlyLeuProLeuLeuLeuProArgLysValGlySerAsp                              505560                                                                        ValGlnLeuPheGlyPheThrValProLysAsnAlaGlnValIleIle                              65707580                                                                      AsnAlaTrpAlaIleGlyArgAspProAspCysTrpGlnLysProAsn                              859095                                                                        SerPheGluProGluArgPheLeuGlySerGlnIleAspValLysGly                              100105110                                                                     ArgAspPheGluLeuIleProPheGlyAlaGlyArgSerIleCysAla                              115120125                                                                     (2) INFORMATION FOR SEQ ID NO:57:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 120 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..120                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                      TTGGAGTGGGCAATGGCAGAACTTCTACGCAACCCGCACACCATGGCC48                            LeuGluTrpAlaMetAlaGluLeuLeuArgAsnProHisThrMetAla                              151015                                                                        AAAGCAAAAGAGGAGCTTAAAGACGTTATCGGCAAAGAAAAACTTGTA96                            LysAlaLysGluGluLeuLysAspValIleGlyLysGluLysLeuVal                              202530                                                                        GATGAAGCTGACATTTTCGAGACT120                                                   AspGluAlaAspIlePheGluThr                                                      3540                                                                          (2) INFORMATION FOR SEQ ID NO:58:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                      LeuGluTrpAlaMetAlaGluLeuLeuArgAsnProHisThrMetAla                              151015                                                                        LysAlaLysGluGluLeuLysAspValIleGlyLysGluLysLeuVal                              202530                                                                        AspGluAlaAspIlePheGluThr                                                      3540                                                                          __________________________________________________________________________

We claim:
 1. An isolated nucleic acid encoding an enzyme havingflavonoid 3'-hydroxylase activity and capable of hydroxylatingdihydrokaempferol (DHK), said isolated nucleic acid selected from thegroup consisting of:(a) an isolated nucleic acid having the nucleic acidsequence substantially as set forth in SEQ ID NO: 49; (b) a nucleotidesequence capable of hybridizing to the nucleotide sequence of SEQ ID NO:49, or a sequence complementary to SEQ ID NO: 49, under hybridizationwashing conditions of 6×SSC and 1% w/v SDS at 65° C.; and (c) anucleotide sequence which is at least 50% identical to the nucleotidesequence of SEQ ID NO:49.
 2. An isolated nucleic acid according to claim1 wherein said isolated nucleic acid encodes an enzyme having an aminoacid sequence substantially as set forth in SEQ ID NO:50.
 3. An isolatednucleic acid according to claim 1 wherein said isolated nucleic acidencodes an enzyme having at least a 29% similarity to SEQ. ID NO:50. 4.An isolated nucleic acid according to any one of claims 1-3 wherein saidisolated nucleic acid is genomic DNA or cDNA.
 5. An isolated nucleicacid according to any one of claims 1-3 wherein said isolated nucleicacid is contained within a plasmid.
 6. An isolated nucleic acidaccording to claim 4 wherein the plasmid is pCGP619.